5ht agonists for treating disorders

ABSTRACT

Provided, inter alia, are methods for treating an epilepsy disorder using a 5HT receptor agonist, or a pharmaceutically acceptable salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 62/120,726filed Feb. 25, 2015, the disclosure of which is incorporated byreference herein in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under grant number R01NS079214 03 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The disclosure provides methods for treating an epilepsy disorder usinga 5HT receptor agonist, or a pharmaceutically acceptable salt thereof.

BACKGROUND OF THE INVENTION

Dravet syndrome (DS) is a catastrophic pediatric epilepsy with severeintellectual disability, impaired social development and persistentdrug-resistant seizures. One of its primary causes is mutations inNav1.1 (SCN1A), a voltage-gated sodium channel. Seizures experienced bythose with DS and other epilepsy disorders are inadequately managedusing available antiepileptic drugs (AEDs) and children with DS are poorcandidates for neurosurgical resection. Thus there is a need in the artfor epilepsy treatment options, especially those for DS and relatedcatastrophic pediatric epilepsies. Provided herein are solutions tothese problems and other problems in the art.

BRIEF SUMMARY OF THE INVENTION

Provided herein, inter alia, are methods of treating epilepsy disordersusing a 5HT agonist, or a pharmaceutically acceptable salt thereof. Inone aspect the method includes administering to a subject in needthereof, a therapeutically effective amount of a 5HT agonist, a 5HTagonist analog, or a pharmaceutically acceptable salt thereof. Inanother aspect the method includes administering to a subject in needthereof, a pharmaceutical composition that includes a therapeuticallyeffective amount of a 5HT agonist, a 5HT agonist analog, or apharmaceutically acceptable salt thereof. Further provided herein arepharmaceutical compositions for treating epilepsy disorders.

According to an aspect of the disclosure, a method of treating anepilepsy disorder may involve administering to a patient having anepilepsy disorder a therapeutically effective amount of a 5HT receptoragonist, or a pharmaceutically acceptable salt thereof.

In an exemplary embodiment, the 5HT agonist may be a 5HT_(2A) receptoragonist or a 5HT_(2B) receptor agonist. In some exemplary embodiment, itis contemplated that the 5HT agonist may be any one or more of ACP-104,ACP-106, AR-116081, AR-116082, ATHX-105, belladonna in combination withergotamine tartrate, BW 723C86, Cisapride, Ciza-MPS, Cizap, Cizap-Mps,CSC-500 Series, DOI or salt thereof (e.g. HCl), Ergotamine Tartrate andCaffeine, Esorid MPS, flibanserin, Ikaran L.P., Manotac Plus, Migril,Mirtazapina Rimafar, mirtazapine, Naratriptan, nelotanserin,norfenfluramine, Normagut Tab, nefazodone hydrochloride, OSU-6162,Pridofin, Sensiflu, PRX-00933, RP-5063, Small Molecule to Agonize5-HT_(2A) for Inflammatory Diseases, Small Molecules to Agonize5-HT_(2C) for Schizophrenia and Obesity, Small Molecules to Agonize5-HT_(2C) Receptor for Obesity, Small Molecules to Target 5-HT_(2C) and5-HT₆ Receptor for Schizophrenia, Small Molecules to Modulate 5HT₂ forCNS and Metabolic Disorders, TGBA-01AD, trazodone hydrochloride,temanogrel hydrochloride, vabicaserin hydrochloride, Virdex, VR-1065,ziprasidone hydrochloride, and/or Ziprasidon-Sigillata.

In some exemplary embodiments, it is contemplated that the 5HT receptoragonist is not clemizole or fenfluramine. In some exemplary embodiments,it is further contemplated that the 5HT receptor agonist is notacetazolamide, benzodiazepine (diazepam; clobazam), cannabadiol,carbamazepine, clemizole, ethosuximide, felbamate, fenfluramine,fluoxetine, gabapentin, ganaxolone, lacosamide, lamotrigine,levetiracetam, nitrazepam, oxcarbazepine, perampenel, phenytoin,phenobarbital, piracetam, potassium bromide, pregabalin, primidone,retigabine, rufinamide, stiripentol, tiagabine, topiramate, valproicacid, verapamil, vigabatrin, and/or zonisamide.

In an exemplary embodiment, the 5HT receptor agonist binds directly to a5HT receptor. In an exemplary embodiment, the 5HT receptor agonistspecifically activates a 5HT receptor.

In an exemplary embodiment, the 5HT receptor agonist provides increasedactivity mediated by 5HT_(2A) receptor or 5HT_(2B) receptor withequivalent or reduced activity mediated through 5HT_(2C) receptor.

In an exemplary embodiment, the 5HT receptor agonist is a dual 5HT_(2A)receptor and 5HT_(2B) receptor agonist.

In an exemplary embodiment, the 5HT receptor agonist is not a serotoninreuptake inhibitor.

In an exemplary embodiment, the 5HT receptor agonist does notsignificantly bind to or modulate the activity of at least one of5HT_(1A), 5HT_(1B), 5HT_(1D), 5HT_(2C), 5HT₃, 5HT₄ (e.g. 5HT_(4e)),5HT₆, 5HT₇, NPY Y1 receptor, L-type Ca channel, N-type Ca channel, SK-Cachannel, GABA-gated C1 channel, GABA transporter, GABA-A1 receptor,GABA-B1b receptor, Na channel, 5HT transporter, CB1 receptor, CB2receptor, BZD or Estrogen ER alpha.

In an exemplary embodiment, the 5HT receptor agonist is any one or moreof flibanserin, 2,5-dimethoxy-4-Iodoamphetamine monohydrochloride (DOIHCl), norfenfluramine or BW 723C86.

In an exemplary embodiment, the epilepsy disorder is Dravet Syndrome,Lennox-Gastaut Syndrome, infantile spasm, or Ohtahara Syndrome. In anexemplary embodiment, the epilepsy disorder is Dravet Syndrome. In anexemplary embodiment, the epilepsy disorder is a pediatric epilepsydisorder.

In an exemplary embodiment, the subject has a cardiovascular disease.

In an exemplary embodiment, the subject is resistant to treatment with aserotonin reuptake inhibitor.

In an exemplary embodiment, the subject is susceptible to side effectswhen administered a serotonin reuptake inhibitor. In an exemplaryembodiment, the serotonin reuptake inhibitor is fenfluramine.

In an exemplary embodiment, the subject has a ketogenic diet.

In an exemplary embodiment, the 5HT receptor agonist inhibits compulsivebehaviors or electrographic seizures in an epilepsy subject, anAlzheimer's disease subject, an autism subject, or a Parkinson's diseasesubject.

In an exemplary embodiment, the 5HT receptor agonist reduces theincidence of unprovoked seizures in said subject when compared to theabsence of said 5HT receptor agonist.

In an exemplary embodiment, the administration of said 5HT receptoragonist reduces or prevents myoclonus seizures or status epilepticus insaid subject when compared to the absence of 5HT receptor agonist.

In an exemplary embodiment, the 5HT receptor agonist is administered tosaid subject at an amount of about 0.1 mg to about 1000 mg per kg bodyweight.

In an exemplary embodiment, the 5HT receptor agonist is administered tosaid subject in a daily dose of about 0.1 mg to about 1000 mg per kgbody weight to said subject.

In an exemplary embodiment, the 5HT receptor agonist is co-administeredwith an anti-epileptic drug (AED). In an exemplary embodiment, the 5HTreceptor agonist is an adjunctive therapy with an anti-epileptic drug(AED). In an exemplary embodiment, the AED is acetazolamide,benzodiazepine, cannabadiols, carbamazepine, clobazam, clonazepam,eslicarbazepine acetate, ethosuximide, ethotoin, felbamate,fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A,lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine,perampanel, piracetam, phenobarbital, phenytoin, potassium bromide,pregabalin, primidone, retigabine, rufinamide, valproic acid, sodiumvalproate, stiripentol, tiagabine, topiramate, vigabatrin, orzonisamide. In an exemplary embodiment, the AED is valproic acid, sodiumvalproate, clonazepam, ethosuximide, felbamate, gabapentin,carbamazepine, oxcarbazepine, lamotrigine, levetiracetam,benzodiazepine, phenobarbital, pregabalin, primidone, tiagabine,topiramate, potassium bromide, phenytoin, stiripentol, vigabatrin, orzonisamide. In an exemplary embodiment, the AED is valproic acid, sodiumvalproate, gabapentin, topiramate, carbamazepine, oxcarbazepine, orvigabatrin.

In an exemplary embodiment, the AED is not a topiramate.

In an exemplary embodiment, the AED is not fenfluramine.

In an exemplary embodiment, the AED is administered simultaneously withor sequentially with said 5HT receptor agonist.

In another aspect, the disclosure provides a method of treating anepilepsy disorder, said method comprising administering to a subject inneed thereof a therapeutically effective amount of a 5HT receptoragonist, or a pharmaceutically acceptable salt thereof, wherein saidsubject has a cardiovascular disease, is resistant to treatment with aserotonin reuptake inhibitor, or is susceptible to side effects whenadministered a serotonin reuptake inhibitor.

In an exemplary embodiment, the 5HT receptor agonist is a 5HT_(2A)receptor agonist or a 5HT_(2B) receptor agonist. In an exemplaryembodiment, the 5HT receptor agonist is clemizole, a clemizole analog,or a pharmaceutically acceptable salt thereof. In an exemplaryembodiment, the pharmaceutically acceptable salt is clemizole HCl.

In embodiments, the 5HT receptor agonist is sumatriptan, naratriptap,rizatriptan, zolmitriptan, urapidil, BRL-54443(3-(1-methylpiperidin-4-yl)-1H-indol-5-ol), lorcaserin, buspirone,ziprasidone, TCB-2((4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide),BRL-15572(3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol),trazodone, BMY 7378(8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione),atomoxetine, and venlafaxine. In embodiments, the 5HT receptor agonistis sumatriptan. In embodiments, the 5HT receptor agonist is naratriptap.In embodiments, the 5HT receptor agonist is rizatriptan. In embodiments,the 5HT receptor agonist is zolmitriptan. In embodiments, the 5HTreceptor agonist is urapidil. In embodiments, the 5HT receptor agonistis BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol). In embodiments,the 5HT receptor agonist is lorcaserin. In embodiments, the 5HT receptoragonist is buspirone. In embodiments, the 5HT receptor agonist isziprasidone. In embodiments, the 5HT receptor agonist is TCB-2((4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide).In embodiments, the 5HT receptor agonist is BRL-15572(3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol). Inembodiments, the 5HT receptor agonist is trazodone. In embodiments, the5HT receptor agonist is BMY 7378(8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione).In embodiments, the 5HT receptor agonist is atomoxetine. In embodiments,the 5HT receptor agonist is venlafaxine.

In embodiments, the 5HT receptor agonist is trazodone or apharmaceutically acceptable salt thereof.

In an exemplary embodiment, the clemizole, said clemizole analog, orsaid pharmaceutically acceptable salt thereof forms part of apharmaceutical composition. In an exemplary embodiment, thepharmaceutical composition further comprises a pharmaceuticallyacceptable excipient. In an exemplary embodiment, the pharmaceuticalcomposition comprises a therapeutically effective amount of clemizole,said clemizole analog, or said pharmaceutically acceptable salt thereof.

In an exemplary embodiment, the pharmaceutical composition isco-administered with an anti-epileptic drug (AED). In an exemplaryembodiment, the pharmaceutical composition comprises clemizole, saidclemizole analog, or said pharmaceutically acceptable salt thereof andan AED. In an exemplary embodiment, the 5HT receptor agonist is notfenfluramine. In an exemplary embodiment, the 5HT receptor agonist bindsdirectly to a 5HT receptor.

In an exemplary embodiment, the 5HT receptor agonist specificallyactivates a 5HT receptor. In an exemplary embodiment, the 5HT receptoragonist provides increased activity mediated by 5HT_(2A) receptor or5HT_(2B) receptor with equivalent or reduced activity mediated through5HT_(2C) receptor. In an exemplary embodiment, the 5HT receptor agonistis a dual 5HT_(2A) receptor and 5HT_(2B) receptor agonist.

In an exemplary embodiment, the 5HT receptor agonist is not a serotoninreuptake inhibitor.

In an exemplary embodiment, the 5HT receptor agonist does notsignificantly bind to at least one of 5HT_(1A), 5HT1_(B), 5HT_(1D),5HT_(2C), 5HT₃, 5HT₄ (e.g. 5HT_(4e)), 5HT₆, 5HT₇, NPY Y1 receptor,L-type Ca channel, N-type Ca channel, SK-Ca channel, GABA-gated C1channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Nachannel, 5HT transporter, CB1 receptor, CB2 receptor, BZD or Estrogen ERalpha.

In another aspect, the disclosure provides a method of modulatingactivity of a 5HT receptor comprising contacting a 5HT receptor withclemizole, a clemizole analog, or a pharmaceutically acceptable saltthereof.

In an exemplary embodiment, the modulating is activating.

In an exemplary embodiment, the 5HT receptor is a 5HT_(2A) receptor or a5HT_(2B) receptor.

In another aspect, the disclosure provides a method of treating adisease or disorder caused by a deficiency of serotonin in the brain orunder activity of one or more 5HT receptors comprising administering toa subject in need thereof a therapeutically effective amount ofclemizole, a clemizole analog, or a pharmaceutically acceptable saltthereof.

In an exemplary embodiment, the disease or disorder is not epilepsy.

In an exemplary embodiment, the disease or disorder is not DravetSyndrome.

In an exemplary embodiment, the disease or disorder is selected from thegroup consisting of migraine, Fragile X syndrome, Prader-Willi syndrome,schizophrenia, depression, Alzheimer's disease, autism, neuropathicpain, Parkinson's disease, irritable bowel disorder, and dementia.

In one aspect there is provided a pharmaceutical composition thatincludes clemizole, a clemizole analog, or a pharmaceutically acceptablesalt thereof for use in treating a disease or disorder caused by adeficiency of serotonin in the brain or under activity of one or more5HT receptors.

In embodiments of an aspect above, the 5HT receptor agonist does notsignificantly bind to or modulate (e.g. inhibit) the activity of atleast one of 5HT1A, 5HT1B, 5HT1D, 5HT3, 5HT4e, GABAA1, GABAB_((1b)),BZD. CB₁, CB₂, GABA-gated C1 channel, SK-Ca channel or GABA transporter.In embodiments, the 5HT receptor agonist does not significantly bind toor modulate (e.g. inhibit) the activity of 5HT1A, 5HT1B, 5HT1D, 5HT3,5HT4e, GABAA1, GABAB_((1b)), BZD. CB₁, CB₂, GABA-gated C1 channel, SK-Cachannel and GABA transporter.

In embodiments of an aspect above, the 5HT receptor agonist modulates atarget less than about 50% relative to a reference modulator (e.g. anatural or known modulator as commonly used in the art). In embodiments,the 5HT receptor agonist modulates a target less than about 40% relativeto a reference modulator (e.g. a natural or known modulator as commonlyused in the art). In embodiments, the 5HT receptor agonist modulates atarget less than about 30% relative to a reference modulator (e.g. anatural or known modulator as commonly used in the art). In embodiments,the 5HT receptor agonist modulates a target less than about 20% relativeto a reference modulator (e.g. a natural or known modulator as commonlyused in the art). In embodiments, the 5HT receptor agonist modulates atarget less than about 10% relative to a reference modulator (e.g. anatural or known modulator as commonly used in the art).

In embodiments of an aspect above, the 5HT receptor agonist does notsignificantly modulate the activity when the 5HT receptor agonist failsto modulate (e.g. inhibit) a target less than about 50% relative to areference inhibitor (e.g. a natural or known inhibitor as commonly usedin the art). In embodiments, the 5HT receptor agonist does notsignificantly modulate the activity when the 5HT receptor agonist failsto modulate (e.g. inhibit) a target less than about 40% relative to areference inhibitor (e.g. a natural or known inhibitor as commonly usedin the art). In embodiments, the 5HT receptor agonist does notsignificantly modulate the activity when the 5HT receptor agonist failsto modulate (e.g. inhibit) a target less than about 30% relative to areference inhibitor (e.g. a natural or known inhibitor as commonly usedin the art). In embodiments, the 5HT receptor agonist does notsignificantly modulate the activity when the 5HT receptor agonist failsto modulate (e.g. inhibit) a target less than about 20% relative to areference inhibitor (e.g. a natural or known inhibitor as commonly usedin the art). In embodiments, the 5HT receptor agonist does notsignificantly modulate the activity when the 5HT receptor agonist failsto modulate (e.g. inhibit) a target less than about 10% relative to areference inhibitor (e.g. a natural or known inhibitor as commonly usedin the art). In embodiments, the 5HT receptor agonist does notsignificantly modulate the activity when the 5HT receptor agonist failsto modulate (e.g. inhibit) a target less than about 5% relative to areference inhibitor (e.g. a natural or known inhibitor as commonly usedin the art).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Molecular characterization of scn1Lab zebrafish mutants. (A)Sequencing confirmed a T-to-G mutation in scn1Lab mutant cDNA. (B)Verification of reduced expression in scn1Lab mutants compared tosibling controls at 3, 5 and 7 dpf using qPCR. Data presented asmean±S.E.M; *significance taken as p<0.05 student's t-test. Data werenormalized to internal reference gene (3-actin. Values representaverages from five independent biological samples (1 sample=10 pooledlarvae) for each of the 3 developmental stages. Data presented asmean±S.E.M; *significance taken as p<0.05 student's t-test. (C) Relativeexpression of scn8aa and scn8ab in Na_(v)1.1 mutants (n=5) and siblingcontrols (n=5) at 5 dpf. Data presented as in B. (D) Whole-mount in situhybridization for scn1Lab in larval zebrafish at 3, 5 and 7 dpf.Wild-type larvae are shown in lateral views; expression is shown in darkpurple. Scn1Laa expression at 3 dpf is shown for comparison. Heartindicated by arrowheads in 5 and 7 dpf panels. (E) Dorsal view ofscb1Laa expression at 3 dpf; note prominent expression in regionscorresponding to the larval zebrafish CNS. Abbreviations: Tel,telencephalon; TeO, optic tectum; Cb, cerebellum. Scale bars=0.35 mm inD, 0.2 mm in E.

FIG. 2. Microarray analysis of scn1Lab zebrafish mutants. (A) Heat mapsdepicting the expression of genes differentially expressed betweenscn1Lab mutant and sibling control larvae at 5 dpf. Rows representindividual genes. Columns represent different larvae. Genes that arehighly expressed in scn1Lab mutants relative to controls are as shown.(B) MA plot of normalized microarray data for all 44,000 genes. Thelog-ratio M and the mean fluorescence intensity A were calculated as theaverages for all replicates. (C) A list of the top 30 genes showing thegreatest differences in expression between scn1Lab mutants and siblingcontrols.

FIG. 3. Quantitative RT-PCR analysis of scn1Lab zebrafish mutants. (A)Comparison of the gene expression fold changes obtained by microarrayanalysis (array) and real-time qPCR analysis. The y-axis represents theaverage fold change in gene expression of each gene from zebrafish at 5dpf. The x-axis represents different genes. (B) qPCR analysis of threegenes involved in epileptogenesis. The relative gene expression ispresented as log₂ ratios to the least abundant transcript (log₂ββct).Data were normalized to internal reference gene β-actin. Valuesrepresent averages from five independent biological samples (1 sample 10pooled larvae). Bars indicate S.E.M; *p<0.05 t-test. (C) Gene ontologyclassification of differentially expressed genes detected in scn1Labmutants at 5 dpf (p<0.05 ANOVA one-way and fold changes >1.5).Biological processes representing at least 5 gene annotations in atleast one category are displayed.

FIG. 4. Spontaneous seizures in scn1Lab zebrafish mutants. (A)Immobilized and agar-embedded zebrafish larvae are shown. Images wereobtained using a 4× objective and 2× magnifier on an Olympus uprightmicroscope during forebrain electrophysiological recordings in siblingcontrol (A, left) and scn1Lab mutant (A, middle) larvae at 5 dpf. Notethe dark pigmentation for mutants. Recording electrodes can be seen inpanels A1-2 and the approximate site of the recording electrode tip inthe forebrain (red circle) is shown using a representative HuC:GFPlabeled larvae in A, right. Scale bar: 100 μm. (B) Sample locomotiontracking plot for sibling control (B, left) and scn1Lab mutant (B,right) larvae at 5 dpf. (C) Representative 10 min recording epochsobtained in the forebrain of paralyzed, immobilized and agar-embeddedscn1Lab mutant larvae between 3 and 7 dpf. Note the presence of smalland large amplitude spontaneous burst discharge; additional temporalexpansions of seizure activity. A representative recording, underidentical recording conditions, from a sibling control larvae at 5 dpfis also shown. Scale bar: 2 mV; 30 sec.

FIG. 5. Pharmacological validation of scn1Lab zebrafish mutants. (A)Heat map showing the response to nine different AEDs. Each columnrepresents the percent change in burst frequency(baseline−drug/baseline×100) for one individual zebrafish mutant. Drugsthat inhibit seizure events are shown in dark blue. All drugs weretested at a concentration of 1 mM. Note in some trials carbamazepine andvigabatrin increased burst frequency over the initial baseline levels.(B) Plot of the mean change in burst frequency and standard error forthe data shown in the heat map. Paired t-test or Wilcoxon signed ranksum test for data that failed the normality test showed significance asfollows: diazepam (p=0.002; n=7), potassium bromide (p=0.016; n=7),stiripentol (p=0.024; n=7), and valproate (p=0.004; n=7). (C) Plot ofthe burst duration for all trials shown in A. Data is presented as themean±S.E.M. for electrographic seizure events at baseline (black bars)and after drug exposure (white bars). Inset shows a representative 2 minrecording during the stiripentol trial; scale bars: large trace 1 mV, 1sec; small trace, 1 mV, 100 msec. (D) Plot of the fractional time spentseizing for all trials shown in A. Data is presented as the mean±S.E.M.for electrographic seizure events at baseline (black bars) and afterdrug exposure (white bars). Student's t-test or Mann-Whitney-Rank sumtest for data that failed the normality test showed significance asfollows: diazepam (p=0.001; n=7); potassium bromide (p=0.043; n=7);stiripentol (p=0.007; n=7) and valproate (p=0.007; n=7 (E) Locomotiontracking plots for 10 individual mutant larvae raised in embryo media(top row) or the ketogenic diet for 48 hr. Plots show swim velocity andlocomotion tracks with darker colors indicative of higher velocities; 10min trials are shown. (F) Representative 10 min extracellular recordingepochs from the same fish shown in E; representative examples areindicated by an * in the locomotion plots. Scale bar: 1 mV, 30 sec.Inset shows burst at higher temporal resolution (indicated by #); scalebar: 1 mV, 100 msec.

FIG. 6. A screen to identify drugs that rescue the scn1Lab mutantepilepsy phenotype. (A) Box plot of mean velocity (in mm/sec) for twoconsecutive recordings of mutant larvae in embryo media. The experimentswere performed by first placing the mutant larvae in embryo media andobtaining a baseline locomotion response, embryo media was then replacedwith new embryo media (to mimic the procedure used for test compounds)and a second locomotion response was obtained. The percent change invelocity from baseline (recording #1) vs. experimental (recording #2) isshown. In the boxplot, the bottom and top of the box represent the 25thpercentile and the 75th percentile, respectively. The line across thebox represents the median value, and the vertical lines encompass theentire range of values. This plot represents normal changes in trackingactivity in the absence of a drug challenge. (B) Plot of the effect ofeleven known antiepileptic drugs on locomotor seizure behavior inscn1Lab mutants at 5 dpf. Phenotype-based assay was performed in a96-well format (for example, see panel 5C1). Bars represent the percentchange in mean velocity comparing a baseline recording of mutant seizureactivity with the same mutant after a drug application. For all drugstudies 6-12 fish were used per experiment. Drugs were tested at aconcentration of 1 mM; diazepam (Dzp; p<0.001), carbamazepine (Carb,p=0.024), ganaxolone (Gan; p=0.003), stiripentol (Stp; p=0.001),valproate (Vpa, p=0.026) and a 48 hr exposure to the ketogenic diet (KD;p=0.003) reduced seizure activity, measured as a change in velocity, bymore than 34% (dotted line in B; represents a fold-change greater thanthe standard deviation in control recordings). Acetazolamide (Acet,p<0.001) and ethosuximide (Etx; p=0.250) increased seizure behavior;levetiracetam (Lev; p=0.243), and lamotrigine (Ltg; p=0.058) had noeffect. (C) Plot of locomotor seizure behavior for scn1Laab mutants at 5dpf for the 320 compounds tested. Colored circles represent positivehits; compounds that decreased activity by 100% were generally toxic;6-12 fish per trial. Arrowhead denotes the first clemizole trial. Notesome compounds increased seizure activity, as expected. (D) Plot of drugre-trials on separate clutches of scn1Lab mutants at 5 dpf; 100 μM perdrug; 10 fish per trial. Abbreviations: Clem, clemizole; Clem+PTZ,clemizole+15 mM PTZ; Clorg, clorgiline; Tolp, tolperisone; Zox,zoxazolamine. Effect of acute clemizole on PTZ-induced seizure behavioris shown for wild-type larvae. Bars represent mean±S.E.M. For panels Band D: Student's paired t-test or Mann-Whitney Rank Sum test withsignificance set at p=0.01 (*) or p<0.001 (**). (E) Sampleelectrophysiology recordings from scn1Lab mutants exposed to clemizolefirst in the locomotion assay (panel D) and then monitored using aforebrain extracellular recording electrode (top trace; ictal-like burstshown in inset). Similar traces are shown for an un-treated Na_(v)1.1mutant (middle trace) and a mutant treated with zoxazolamine (bottomtrace). Analysis of bursting for un-treated mutants (n=3): burstfrequency=1.5±0.3 bursts/min; burst duration=926±414 msec; fractionaltime spent seizing=0.73±0.17% vs. clemizole-treated mutants (n=7): burstfrequency=0.2±0.01 bursts/min; burst duration=154±127 msec; fractionaltime spent seizing=0.03±0.02%; p=0.001 for all comparisons,Kruskal-Wallis ANOVA with a Dunn's pairwise multiple comparison test).Scale bars: large trace 0.5 mV, 10 s; inset 0.5 mV, 100 msec.

FIG. 7: Confirmation of clemizole activity in scn1Laa mutants. (A)Representative 10 min recording epochs obtained in the forebrain ofparalyzed, immobilized and agar-embedded scn1Laa mutant larvae at 6 dpf.Note the presence of small and large amplitude spontaneous burstdischarge. (B) Locomotion tracking plots for 10 individual mutant andwild-type sibling larvae. Plots show swim velocity and locomotion trackswith darker colors indicative of higher velocities; 10 min trials areshown. Seizures were scored on a staging system described in Baraban etal. (Neuroscience 2005). S0, little or no swim activity; S1, increasedlocomotion; S2 whirlpool like swim activity and S3; full bodyconvulsions with rapid swimming events and loss of posture. (C) Box plotof mean velocity (in mm/sec) for 96 zebrafish with fish sorted intoputative scn1Laa and sibling control pools based on seizure stagesdescribed above. The experiments were performed by first placing themutant larvae in embryo media and obtaining a baseline locomotionresponse, embryo media was then replaced with new embryo media and asecond locomotion response was obtained. The percent change in velocityfrom baseline (recording #1) vs. experimental (recording #2) is shown.In the boxplot, the bottom and top of the box represent the 25thpercentile and the 75th percentile, respectively. The line across thebox represents the median value, and the vertical lines encompass theentire range of values. Plots are shown for all 96 fish (left), putativescn1Laa zebrafish (middle) and sibling controls (left). Subsequent PCRanalysis was done to confirm mutant and control pools. (D) Plot of theeffect of stiripentol (Stp), diazepam (Dzp), clemizole (Clem) andlamotrigine (Ltg) on locomotor seizure behavior in scn1Laa mutants at 5dpf. Mean velocity is shown before and after application of a drug. N=7fish per drug. Bars represent mean±S.E.M. Student's paired t-test orMann-Whitney Rank Sum test with significance set at p=0.01 (*) orp<0.001 (**).

FIG. 8: Antihistamines do not have antiepileptic properties in scn1Labmutants. Plot of the effect of a variety of antihistamines in thelocomotor seizure assay using scn1Lab mutants at 5 dpf. Mean velocity isshown before and after application of a drug. N=7 fish per drug.Additional compounds are listed at right. Bars represent mean±S.E.M.Student's paired t-test or Mann-Whitney Rank Sum test with significanceset at p=0.01 (*) or p<0.001 (**). Note: some antihistamines increasedseizure activity in this assay.

FIG. 9: Clemizole concentration-response studies in scn1Lab. Plots fromtwo different concentration-response studies showing the percentinhibition of mean velocity from a baseline value. N=7 fish perconcentration and trials were performed on separate clutches of mutantlarvae.

FIG. 10: A shelf screen was performed using 34 different compounds withantihistamine properties. All compounds shown were tested againstspontaneous seizures in scn1Lab larvae at 5 dpf; 6-10 fish per drug.Results are shown as a change in mean velocity from locomotion trackingdata plots. Compounds were tested at concentrations between 0.1 and 1mM. The threshold for a positive hit is indicated by the dashed line.Three compounds reached this threshold, but all 3 were identified astoxic (arrows).

FIG. 11: 5HT library screen. Plot of locomotor seizure behavior forscn1Lab mutants at 5 dpf for the 62 compounds tested. Threshold forinhibition of seizure activity (positive hits) was set as a reduction inmean swim velocity ≧38%; threshold for a proconvulsant or hyperexcitableeffect was set at as an increase in mean swim velocity ≧44% (greendashed lines). Compounds were tested at a concentration of 250 μM and 6fish per drug. Compound list is shown below with positive hits indicatedin grey (on chart) or black circles (in plot).

FIGS. 12A-12B: FIG. 12A. Plot of the change in mean velocity for scn1Labmutants at 5 dpf exposed to different concentrations of trazodone for 30min (black bars) or 90 (gray bars) drug exposure periods. Threshold forinhibition of seizure activity (positive hits) was set as a reduction inmean swim velocity ≧40%. Trazodone was toxic at 750 μM (hashed lines).Compounds were tested at 6 fish per drug. FIG. 12B. Sample EEG tracesfor scn1Lab mutant larvae exposed to trazodone (suppression of epilepticseizure events) or a control drug, MK-801 (no suppression of epilepticevents).

DETAILED DESCRIPTION OF THE INVENTION

By “5HT Receptor” or “5-Hydroxytryptamine Receptor” is meant a group ofG protein-coupled receptors (GPCRs) and ligand-gated ion channels(LGICs) that are found in the central (CNS) and peripheral nervoussystems (PNS) and belong generally to the group of serotonin receptors.5HT receptors may be divided into seven receptor families, including:5HT₁ (e.g., G_(i)/G₀-protein coupled receptors), 5HT₂ (e.g.,G_(q)/G₁₁-protein coupled receptors), 5HT₃ (e.g., ligand-gated Na⁺ andK⁺ cation channel), 5HT₄ (e.g., G₅-protein coupled receptors), 5HT₅(e.g., G_(i)/G₀-protein coupled receptors), 5HT₆ (e.g., G₅-proteincoupled receptors), and 5HT₇ (e.g., G₅-protein coupled receptors).Additionally, the seven families of 5HT receptors may be furthersubdivided into a number of sub-families. For example, the 5HT₁ familyalso includes the following sub-families: 5HT_(1A) (e.g., which areknown to function in blood vessels and the CNS and may be involved inaddiction, aggression, anxiety, appetite, autoreceptor, blood pressure,cardiovascular function, emesis, heart rate, impulsivity, memory, mood,nausea, nociception, penile erection, pupil dilation, respiration,sexual behavior, sleep, sociability, thermoregulation, andvasoconstriction), 5HT_(1B) (e.g., which are known to function in bloodvessels and the CNS and may be involved in addiction, aggression,anxiety, auto receptor, learning, locomotion, memory, mood, penileerection, sexual behavior, and vasoconstriction), 5HT_(1D) (e.g., whichare known to function in blood vessels and the CNS and may be involvedin anxiety, autoreceptor, locomotion, and vasoconstriction), 5HT_(1E)(e.g., which are known to function in the CNS and may be involved inmigraines). As another example, the 5HT₂ family may be divided into thefollowing sub-families: 5HT_(2A) (e.g., which are known to function inblood vessels, CNS, gastrointestinal tract, platelets, PNS, and smoothmuscle, and may be involved in addiction, anxiety, appetite, cognition,imagination, learning, memory, mood, perception, sexual behavior, sleep,thermoregulation, and vasoconstriction), 5HT_(2B) (e.g., which are knownto function in blood vessels, CNS, gastrointestinal tract, platelets,PNS, and smooth muscle, and may be involved in anxiety, appetite,cardiovascular function, gastrointestinal motility, sleep, andvasoconstriction), and 5HT_(2C) (e.g., which are known to function inblood vessels, CNS, gastrointestinal tract, platelets, PNS, and smoothmuscle, and may be involved in addiction, anxiety, appetite,gastrointestinal motility, locomotion, mood, penile erection, sexualbehavior, sleep, thermoregulation, and vasoconstriction). Additionally,the 5HT₅ family may be further divided into the following sub-families:5HT_(5A) (e.g., which may function in the CNS, and play a role inlocomotion and sleep, as well as function as an autoreceptor) and5HT_(5B) (e.g., which may function in rodents and appears to be apseudogene in humans).

By “5HT Receptor agonist” is meant any agent that activates a 5HTreceptor relative to the absence of the 5HT Receptor agonist or in amanner similar to serotonin. Exemplary 5HT receptor agonists include,but are not limited to, any one or more of the following: ACP-104,ACP-106, AR-116081, AR-116082, ATHX-105, belladonna in combination withergotamine tartrate, BW 723C86, Cisapride, Ciza-MPS, Cizap, Cizap-Mps,CSC-500 Series, DOI or salt thereof, Ergotamine Tartrate and Caffeine,Esorid MPS, flibanserin, Ikaran L.P., Manotac Plus, Migril, MirtazapinaRimafar, mirtazapine, Naratriptan, nelotanserin, norfenfluramine,Normagut Tab, nefazodone hydrochloride, OSU-6162, Pridofin, Sensiflu,PRX-00933, RP-5063, Small Molecule to Agonize 5-HT_(2A) for InflammatoryDiseases, Small Molecules to Agonize 5-HT_(2C) for Schizophrenia andObesity, Small Molecules to Agonize 5-HT_(2C) Receptor for Obesity,Small Molecules to Target 5-HT_(2C) and 5-HT₆ Receptor forSchizophrenia, Small Molecules to Modulate 5HT₂ for CNS and MetabolicDisorders, TGBA-01AD, trazodone hydrochloride, temanogrel hydrochloride,vabicaserin hydrochloride, Virdex, VR-1065, ziprasidone hydrochloride,and Ziprasidon-Sigillata.

In embodiments, the 5HT receptor agonist is sumatriptan, naratriptap,rizatriptan, zolmitriptan, urapidil, BRL-54443(3-(1-methylpiperidin-4-yl)-1H-indol-5-ol), lorcaserin, buspirone,ziprasidone, TCB-2((4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide),BRL-15572(3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol),trazodone, BMY 7378(8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione),atomoxetine, or venlafaxine.

In embodiments, the 5HT receptor agonist is trazodone.

It is further contemplated within the scope of the disclosure that insome embodiments, a 5HT Receptor agonist does not include one or more orall of the following: acetazolamide, benzodiazepine (diazepam;clobazam), cannabadiol, carbamazepine, clemizole, ethosuximide,felbamate, fenfluramine, fluoxetine, gabapentin, ganaxolone, lacosamide,lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampenel,phenytoin, phenobarbital, piracetam, potassium bromide, pregabalin,primidone, retigabine, rufinamide, stiripentol, tiagabine, topiramate,valproic acid, verapamil, vigabatrin, and zonisamide.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “cardiac condition” is meant related diseases, including, but notlimited to: coronary heart disease (CHD), cardiomyopathy, cardiovasculardisease (CVD), ischemic heart disease, heart failure, hypertensive heartdisease, inflammatory heart disease, valvular heart disease,atherosclerosis, and cardiac hypertrophy. A cardiac condition may be asystemic disease that can affect the heart, brain, most major organs,and the extremities. By “coronary heart disease (CHD)” is meant adisease that causes the failure of coronary circulation to supplyadequate circulation to the cardiac muscles and surrounding tissues. By“cardiovascular disease (CVD)” is meant any of a number of specificdiseases that affect the heart itself or the blood vessel system,especially the myocardial tissue, as well as veins and arteries leadingto and from the heart. For example, CVD may include, but is not limitedto, acute coronary syndromes, arrhythmia, atherosclerosis, heartfailure, myocardial infarction, neointimal hyperplasia, pulmonaryhypertension, stroke, valvular disease, or cardiac hypertrophy. Heartdisease may be diagnosed by any of a variety of methods known in theart. For example, such methods may include assessing a subject fordyspnea, orthopnea, paroxysmal nocturnal dyspnea, claudication, angina,chest pain, which may present as any of a number of symptoms known inthe art, such as exercise intolerance, edema, palpitations, faintness,loss of consciousness, or cough; heart disease may be diagnosed by bloodchemistry analysis. As described above and as used herein, “heartdisease” relates to a disorder affecting the heart itself or thecirculatory system.

“Analog” or “analogue” is used in accordance with its plain ordinarymeaning within Chemistry and Biology and refers to a chemical compoundthat is structurally similar to another compound (i.e., a so-called“reference” compound) but differs in composition, e.g., in thereplacement of one atom by an atom of a different element, or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group, or the absolutestereochemistry of one or more chiral centers of the reference compound.Accordingly, an analog is a compound that is similar or comparable infunction and appearance but not in structure or origin to a referencecompound.

“Clemizole” refers to a compound having formula:

Clemizole includes pharmaceutically acceptable salts and formulations ofclemizole as described herein (e.g. a “clemizole salt”). Exemplary,clemizole salts include but are not limited to clemizole-HCl,clemizolpenicillin, clemizole-sulfate, or clemizole-undecylate.

A “clemizole analog” as set forth herein refers to compounds of similarstructure. Such compounds include, for example, those compounds setforth in PCT/US2008/076804, and U.S. Pat. No. 4,011,322, which areherein incorporated by reference in their entirety. Further exemplaryclemizole analogs are set forth, for example in: US 2012/0232062; PCTPub. Nos. 2009/038248; US 2010/107739; US 2010/107742, WO 2002/089731.WO 2005/032329, WO 2009/039248, WO 2010/039195, WO 2010/107739, and WO2010/107742, each of which is incorporated herein by reference in theirentirety. Clemizole analogs described herein (including the compoundsdescribed in the references above) may be substituted (i.e. modified) atthe 1 or 2 position as set forth below in formula (I) (boxes Y and Z).Clemizole analogs may be substituted (i.e. modified) at 4, 5, 6, or 7positions as indicated by box X in formula (I).

“Flibanserin” refers to a compound having the following formula (II):

Flibanserin includes pharmaceutically acceptable salts and formulationsof flibanserin (e.g. a “flibanserin salt”).

“Norfenfluramine” refers to a compound having the following formula(III):

Norfenfluramine includes pharmaceutically acceptable salts andformulations of norfenfluramine (e.g. a “norfenfluramine salt”).

“DOI” refers to 2,5-dimethoxy-4-Iodoamphetamine. DOI includespharmaceutically acceptable salts and formulations of DOI. Exemplary,DOI salts include but are not limited to 2,5-dimethoxy-4-Iodoamphetaminemonohydrochloride (DOI HCl) having the following formula (IV):

“BW 723C86” refers to a tryptamine derivative drug that acts as a5HT_(2B) receptor agonist and has the following formula (V):

BW 723C86 includes pharmaceutically acceptable salts and formulations ofBW 723C86 (e.g. a “BW 723C86 salt”).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When the 5-HT agonist contains relativelyacidic functionalities, base addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When the 5-HT agonist contains relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977,66, 1-19). The 5-HT agonist may contain both basic and acidicfunctionalities that allow conversion into either base or acid additionsalts.

Thus, the 5-HT agonist may exist as a salt, such as withpharmaceutically acceptable acids. The present invention includes suchsalts. Non-limiting examples of such salts include hydrochlorides,hydrobromides, phosphates, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g.,(+)-tartrates, (−)-tartrates, or mixtures thereof including racemicmixtures), succinates, benzoates, and salts with amino acids such asglutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyliodide, and the like). These salts may be prepared by methods known tothose skilled in the art.

The neutral forms of the 5-HT agonist are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compound maydiffer from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the 5-HT agonist may be provided in a prodrugform. Prodrugs are those compounds that readily undergo chemical changesunder physiological conditions to provide the compounds of the presentinvention. Prodrugs of the 5-HT agonist may be converted in vivo afteradministration. Additionally, prodrugs of the 5-HT agonist can beconverted to active compounds by chemical or biochemical methods in anex vivo environment, such as, for example, when contacted with asuitable enzyme or chemical reagent.

The 5-HT agonist can exist in unsolvated forms as well as solvatedforms, including hydrated forms. In general, the solvated forms areequivalent to unsolvated forms and are encompassed within the scope ofthe present invention. The 5-HT agonist may exist in multiplecrystalline or amorphous forms. In general, all physical forms areequivalent for the uses contemplated by the present invention and areintended to be within the scope of the present invention.

An “effective amount” is an amount sufficient for the 5-HT agonist(including pharmaceutically acceptable salts thereof) to accomplish astated purpose relative to the absence of a 5-HT agonist (includingpharmaceutically acceptable salts thereof) (e.g. achieve the effect forwhich it is administered, treat a disease, reduce protein/enzymeactivity, increase protein/enzyme activity, reduce a signaling pathway,or reduce one or more symptoms of a disease or condition). An example ofan “effective amount” is an amount of the 5-HT agonist (includingpharmaceutically acceptable salts thereof) which is sufficient tocontribute to the treatment, prevention, or reduction of a symptom orsymptoms of a disease, which could also be referred to as a“therapeutically effective amount.” A “reduction” of a symptom orsymptoms (and grammatical equivalents of this phrase) means decreasingof the severity or frequency of the symptom(s), or elimination of thesymptom(s) (e.g. seizures). A “prophylactically effective amount” of adrug is an amount of a drug that, when administered to a subject, willhave the intended prophylactic effect, e.g., preventing or delaying theonset (or reoccurrence) of an injury, disease, pathology or condition,or reducing the likelihood of the onset (or reoccurrence) of an injury,disease, pathology, or condition, or their symptoms (e.g. seizures). Thefull prophylactic effect does not necessarily occur by administration ofone dose, and may occur only after administration of a series of doses.Thus, a prophylactically effective amount may be administered in one ormore administrations. The exact amounts will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

The therapeutically effective amount of the 5-HT agonist (includingpharmaceutically acceptable salts thereof) can be initially determinedfrom cell culture assays. Target concentrations will be thoseconcentrations of active compound(s) that are capable of achieving themethods described herein, as measured using the methods described hereinor known in the art.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring compounds effectiveness and adjusting the dosage upwardsor downwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In some embodiments, acontrol is the measurement of the activity of a 5-HT agonist in theabsence of a 5-HT agonist (including pharmaceutically acceptable saltsthereof).

A “test compound” as used herein refers to an experimental compound usedin a screening process to identify activity, non-activity, or othermodulation of a particularized biological target or pathway, for examplea 5-HT receptor. A test compound may be a 5-HT agonist described herein,including pharmaceutically acceptable salts thereof.

The term “modulation”, “modulate”, or “modulator” are used in accordancewith their plain ordinary meaning and refer to the act of changing orvarying one or more properties. “Modulator” refers to a composition orcompound that increases or decreases the level of a target molecule orthe function of a target molecule or the physical state of the target ofthe molecule. “Modulation” refers to the process of changing or varyingone or more properties. For example, as applied to the effects of amodulator on a biological target, to modulate means to change byincreasing or decreasing a property or function of the biological targetor the amount of the biological target (e.g. a 5-HT receptor).

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor interaction meansnegatively affecting (e.g. decreasing) the activity or function of theprotein relative to the activity or function of the protein in theabsence of the inhibitor. In some embodiments inhibition refers toreduction of a disease or symptoms of disease. In some embodiments,inhibition refers to a reduction in the activity of a particular proteinor nucleic acid target. Thus, inhibition includes, at least in part,partially or totally blocking stimulation, decreasing, preventing, ordelaying activation, or inactivating, desensitizing, or down-regulatingsignal transduction or protein/enzymatic activity or the amount of aprotein.

The term “activation” or “activating” and the like in reference toprotein-compound interactions that positively affect (e.g. increase) theactivity or function of the protein relative to the activity or functionof the protein in absence of the activator compound. Activation mayrefer to enhanced activity of a particular protein target. Activationmay refer to restoration of loss-of-function of a mutated proteintarget. Activation as used herein may refer to activation of one or more5-HT receptors.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents that can be producedin the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be a compoundas described herein and a protein or enzyme. In some embodimentscontacting includes allowing a compound described herein to interactwith a receptor, e.g. a 5-HT receptor.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease means thatthe disease is caused by (in whole or in part), or a symptom of thedisease is caused by (in whole or in part) the substance or substanceactivity or function.

The term “patient” or “subject in need thereof” refers to a livingorganism suffering from or prone to a disease or condition that can betreated by administration of a pharmaceutical composition as providedherein. Non-limiting examples include humans, other mammals, bovines,rats, mice, dogs, monkeys, goat, sheep, cows, deer, and othernon-mammalian animals such as zebrafish. A patient may be human.

The term “disease” or “condition” refer to a state of being or healthstatus of a patient or subject capable of being treated with thecompounds or methods provided herein.

The terms “epileptic disorder,” “epilepsy disorder,” “seizure disorder,”or “epilepsy” herein refer to a spectrum of chronic neurologicaldisorders most often characterized by the presence of unprovokedseizures. See e.g. Noebels et. al., Jasper's Basic Mechanisms of theEpilepsies, 4th edition, Bethesda (Md.): National Center forBiotechnology Information (US); 2012. Epilepsy as used herein, may referto injury to the brain (e.g. from trauma, stroke, or cancer) or geneticmutation. The symptoms of epilepsy disorders may result from abnormalelectrochemical signaling between neurons in the brain. Patientsexperiencing two or more unprovoked seizures may be considered to haveepilepsy.

Types of epilepsy disorders include, for example, benign Rolandicepilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonicepilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy(e.g. pyknolepsy), febrile seizures, progressive myoclonus epilepsy ofLafora, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Dravetsyndrome (DS), Generalized Epilepsy with Febrile Seizures (GEFS+),Severe Myoclonic Epilepsy of Infancy (SMEI), Benign Neonatal FamilialConvulsions (BFNC), West Syndrome, Ohtahara Syndrome, early myoclonicencephalopathies, migrating partial epilepsy, infantile epilepticencephalopathies, Tuberous Sclerosis Complex (TSC), focal corticaldysplasia, Type I Lissencephaly, Miller-Dieker Syndrome, Angelman'ssyndrome, Fragile X syndrome, epilepsy in autism spectrum disorders,subcortical band heterotopia, Walker-Warburg syndrome, Alzheimer'sdisease, posttraumatic epilepsy, progressive myoclonus epilepsies,reflex epilepsy, Rasmussen's syndrome, temporal lobe epilepsy, limbicepilepsy, status epilepticus, abdominal epilepsy, massive bilateralmyoclonus, catamenial epilepsy, Jacksonian seizure disorder,Unverricht-Lundborg disease, or photosensitive epilepsy.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” or “carrier moiety” refer to a substance that aids theadministration of the 5-HT agonist (including pharmaceuticallyacceptable salts thereof) to and absorption by a subject and can beincluded in the compositions without causing a significant adversetoxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutically acceptable excipientsare useful in the present invention.

The term “preparation” is intended to include the formulation of the5-HT agonist (including pharmaceutically acceptable salts thereof) withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intrathecal, intranasalor subcutaneous administration, or the implantation of a slow-releasedevice, e.g., a mini-osmotic pump, to a subject. Administration is byany route, including parenteral and transmucosal (e.g., buccal,sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).Parenteral administration includes, e.g., intravenous, intramuscular,intra-arteriole, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial. Other modes of delivery include, butare not limited to, the use of liposomal formulations, intravenousinfusion, transdermal patches, etc.

The 5-HT agonist (including pharmaceutically acceptable salts thereof)and pharmaceutical compositions thereof can be delivered transdermally,by a topical route, formulated as applicator sticks, solutions,suspensions, emulsions, gels, creams, ointments, pastes, jellies,paints, powders, and aerosols. Oral preparations include tablets, pills,powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups,slurries, suspensions, etc., suitable for ingestion by the patient.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. Liquid formpreparations include solutions, suspensions, and emulsions, for example,water or water/propylene glycol solutions. The 5-HT agonists (includingpharmaceutically acceptable salts thereof) may additionally includecomponents to provide sustained release and/or comfort. Such componentsinclude high molecular weight, anionic mucomimetic polymers, gellingpolysaccharides and finely-divided drug carrier substrates. Thesecomponents are discussed in greater detail in U.S. Pat. Nos. 4,911,920;5,403,841; 5,212,162; and 4,861,760. The entire contents of thesepatents are incorporated herein by reference in their entirety for allpurposes. The 5-HT agonist (including pharmaceutically acceptable saltsthereof) can also be delivered as microspheres for slow release in thebody. For example, microspheres can be administered via intradermalinjection of drug-containing microspheres, which slowly releasesubcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; asbiodegradable and injectable gel formulations (see, e.g., Gao Pharm.Res. 12:857-863, 1995); or, as microspheres for oral administration(see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). Theformulations of the compositions of the 5-HT agonist (includingpharmaceutically acceptable salts thereof) can be delivered by the useof liposomes which fuse with the cellular membrane or are endocytosed,i.e., by employing receptor ligands attached to the liposome, that bindto surface membrane protein receptors of the cell resulting inendocytosis. By using liposomes, particularly where the liposome surfacecarries receptor ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the compositions of the 5-HT agonist (including pharmaceuticallyacceptable salts thereof) into the target cells in vivo. (See, e.g.,Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin.Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587,1989). The compositions can also be delivered as nanoparticles.

By “co-administer” it is meant that a composition described herein isadministered at the same time, just prior to, or just after theadministration of one or more additional therapies. The 5-HT agonist(including pharmaceutically acceptable salts thereof) can beadministered alone or can be co-administered to the patient.Co-administration is meant to include simultaneous or sequentialadministration of the compounds individually or in combination (morethan one compound). Thus, the preparations can also be combined, whendesired, with other active substances (e.g. to reduce metabolicdegradation). The 5-HT agonist (including pharmaceutically acceptablesalts thereof) can be delivered transdermally, by a topical route, orformulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

The terms “add on therapy,” “add-on therapy,” “adjunct therapy,” and“adjunctive therapy” are used interchangeably herein and refer tocombining of the 5-HT agonist or a pharmaceutically acceptable saltthereof with another anticonvulsant to treat epilepsy.

An “anti-seizure drug”, “anti-epilepsy drug”, “AED” or “anticonvulsant”are used interchangeably herein and according to their common andordinary meaning and include compositions for reducing or eliminatingseizures. Anticonvulsants include, but are not limited to acetazolamide,benzodiazepine, cannabadiols, carbamazepine, clobazam, clonazepam,eslicarbazepine acetate, ethosuximide, ethotoin, felbamate,fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A,lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine,perampanel, piracetam, phenobarbital, phenytoin, potassium bromide,pregabalin, primidone, retigabine, rufinamide, valproic acid, sodiumvalproate, stiripentol, tiagabine, topiramate, vigabatrin, orzonisamide.

As used herein, the term “resistant” refers to a reduction in theeffectiveness of an agent or drug. For example, a subject that is“resistant” to treatment with a serotonin reuptake inhibitor (such as,e.g. fenfluramine) displays a reduced response relative to the responseobserved when the subject was first treated with the serotonin reuptakeinhibitor.

As used herein, the term “does not significantly bind” shall be taken tomean that a 5HT Receptor agonist of the disclosure displays 10 fold, or20 fold or 50 fold or 60 fold or 70 fold or 80 fold or 90 fold or 100fold, less binding to another transmembrane protein, such as, forexample, a 5HT receptor, a NPY YP receptor, a Ca⁺ channel, a C1 channel,a GABA transporter or GABA-A1 receptor, a Na channel, a 5HT transporter,and/or a CB1 or CB2 receptor. This decreased level of binding may bemeasured by ELISA or biosensor analysis (e.g., Biacore). In one example,a 5HT Receptor agonist of the disclosure does not detectably bind to5HT_(1A), 5HT_(1B), 5HT_(1D), 5HT_(2C), 5HT₃, 5HT₄, 5HT₆, 5HT₇, NPY Y1receptor, L-type Ca channel, Ca channel (N-type), SK-Ca channel,GABA-gated C1 channel, GABA transporter, GABA-A1 receptor, GABA-B1breceptor, Na channel, 5HT transporter, CB1 receptor, CB2 receptor, BZDor Estrogen ER alpha, as measured by ELISA or Biacore or Western Blot orFACS. In this regard, “does not detectably bind” shall be understood tomean that the level of binding is not significantly greater thanbackground. In exemplary embodiments, the 5-HT agonist does notsignificantly modulate the activity of at least one of 5HT_(1A),5HT_(1B), 5HT_(1D), 5HT_(2C), 5HT₃, 5HT₄, 5HT₆, 5HT₇, NPY Y1 receptor,L-type Ca channel, N-type Ca channel, SK-Ca channel, GABA-gated C1channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Nachannel, 5HT transporter, CB1 receptor, CB2 receptor, BZD or Estrogen ERalpha. As used herein, the term “does not significantly modulate theactivity” shall be taken to mean that a 5HT Receptor agonist of thedisclosure elicits a receptor mediated biological response which issubstantially similar to its activity absent activating or inhibitingligand.

Methods of Treatment

Provided herein are methods of treating an epilepsy disorder. In oneaspect, the method is a method of treating an epilepsy disorder byadministering to a subject in need thereof, a therapeutically effectiveamount of a 5-HT receptor agonist or a pharmaceutically acceptable saltthereof. In another aspect, the method is a method of treating anepilepsy disorder by administering to a subject in need thereof, apharmaceutical composition as described herein, where the pharmaceuticalcomposition includes a 5-HT receptor agonist or a pharmaceuticallyacceptable salt thereof. The subject may have (e.g. may eat food inaccordance with) a ketogenic diet. The subject may have a cardiovasculardisease. The subject may be resistant to treatment with a serotoninreuptake inhibitor. The subject may be susceptible to side effects whenadministered a serotonin reuptake inhibitor. The subject may be a child(e.g. a subject having a pediatric epilepsy condition).

The epilepsy disorder may be benign Rolandic epilepsy, frontal lobeepilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenileabsence epilepsy, childhood absence epilepsy (e.g. pyknolepsy), febrileseizures, progressive myoclonus epilepsy of Lafora, Lennox-Gastautsyndrome, Landau-Kleffner syndrome, Dravet syndrome, GeneralizedEpilepsy with Febrile Seizures (GEFS+), Severe Myoclonic Epilepsy ofInfancy (SMEI), Benign Neonatal Familial Convulsions (BFNC), WestSyndrome, Ohtahara Syndrome, early myoclonic encephalopathies, migratingpartial epilepsy, infantile epileptic encephalopathies, TuberousSclerosis Complex (TSC), focal cortical dysplasia, Type I Lissencephaly,Miller-Dieker Syndrome, Angelman's syndrome, Fragile X syndrome,epilepsy in autism spectrum disorders, subcortical band heterotopia,Walker-Warburg syndrome, Alzheimer's disease, posttraumatic epilepsy,progressive myoclonus epilepsies, reflex epilepsy, Rasmussen's syndrome,temporal lobe epilepsy, limbic epilepsy, status epilepticus, abdominalepilepsy, massive bilateral myoclonus, catamenial epilepsy, Jacksonianseizure disorder, Unverricht-Lundborg disease, or photosensitiveepilepsy. The epilepsy may include generalized seizures or partial (i.e.focal) seizures.

The epilepsy disorder may be Dravet Syndrome, Lennox-Gastaut Syndrome,infantile spasm, or Ohtahara Syndrome. The epilepsy disorder may beDravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, or OhtaharaSyndrome, or a pediatric epilepsy disorder. The pediatric epilepsydisorder may be benign childhood epilepsy, Benign Neonatal FamilialConvulsions (BFNC), febrile seizures, Dravet Syndrome, Lennox-GastautSyndrome, infantile spasm, Ohtahara Syndrome, juvenile myoclonicepilepsy, juvenile absence epilepsy, childhood absence epilepsy (e.g.pyknolepsy), infantile spasms. The epilepsy disorder may be DravetSyndrome.

The pediatric epilepsy disorder may be benign childhood epilepsy. Thepediatric epilepsy disorder may be Benign Neonatal Familial Convulsions(BFNC). The pediatric epilepsy disorder may be febrile seizures. Thepediatric epilepsy disorder may be Dravet Syndrome. The pediatricepilepsy disorder may be Lennox-Gastaut Syndrome. The pediatric epilepsydisorder may be infantile spasm. The pediatric epilepsy disorder may beOhtahara Syndrome. The pediatric epilepsy disorder may be juvenilemyoclonic epilepsy. The pediatric epilepsy disorder may be juvenileabsence epilepsy. The pediatric epilepsy disorder may be childhoodabsence epilepsy (e.g. pyknolepsy). The pediatric epilepsy disorder maybe infantile spasms.

The epilepsy disorder may be a result of a neurological disease orinjury such as, for example, encephalitis, cerebritis, abscess, stroke,tumor, trauma, genetic, tuberous sclerosis, cerebral dysgenesis, orhypoxic-ischemic encephalophathy. The epilepsy disorder may beassociated with a neurodegenerative disease such as, for example,Alzheimer's disease or Parkinson's Disease. The epilepsy disorder may beassociated with autism. The epilepsy disorder may be associated with asingle gene mutation. The epilepsy disease may be associated withcompulsive behaviors or electrographic seizures. The administration ofthe 5-HT receptor agonist or a pharmaceutical acceptable salt thereofmay inhibit compulsive behaviors or electrographic seizures in aepilepsy disorder, in an Alzheimer's disease subject (e.g. a subjectsuffering from Alzheimer's disease), in an autism subject (e.g. asubject having autism), or in a Parkinson's disease subject (e.g. asubject suffering from Parkinson's disease). Thus, the 5-HT receptoragonist or a pharmaceutical acceptable salt thereof may inhibitcompulsive behaviors or electrographic seizures in a epilepsy disorder.The 5-HT agonist or a pharmaceutical acceptable salt thereof may inhibitcompulsive behaviors or electrographic seizures in an Alzheimer'sdisease subject. The 5-HT receptor agonist or a pharmaceuticalacceptable salt thereof may inhibit compulsive behaviors orelectrographic seizures in an autism subject. The 5-HT receptor agonistor a pharmaceutical acceptable salt thereof may inhibit compulsivebehaviors or electrographic seizures in a Parkinson's disease subject.

The administration of the 5-HT receptor agonist or a pharmaceuticalacceptable salt thereof may reduce the incidence (e.g. number ofoccurrences) of unprovoked seizures in the subject compared to theabsence of a 5-HT receptor agonist or the pharmaceutically acceptablesalt thereof. Thus, a patient's response to the administration of the5-HT receptor agonist or a pharmaceutical acceptable salt thereof, maybe monitored progressively compared to a time before the administrationof compounds described herein (e.g. a control or control time).

The administration of the 5-HT receptor agonist or a pharmaceuticalacceptable salt thereof may reduce or prevent myoclonus seizures orstatus epilepticus in the subject compared to the absence of a 5-HTreceptor agonist or the pharmaceutically acceptable salt thereof. Theadministration of the 5-HT receptor agonist or a pharmaceuticalacceptable salt thereof may reduce or prevent myoclonus seizures in thesubject compared to the absence of a 5-HT receptor agonist or thepharmaceutically acceptable salt thereof. The administration of the 5-HTreceptor agonist or a pharmaceutical acceptable salt thereof may reduceor prevent status epilepticus in the subject compared to the absence ofa 5-HT receptor agonist or the pharmaceutically acceptable salt thereof.Thus, a patient's response to the administration of the 5-HT receptoragonist or a pharmaceutical acceptable salt thereof, may be monitoredprogressively compared to a time before the administration of compoundsdescribed herein (e.g. a control or control time).

The epilepsy disorder may be an epilepsy disorder which isnon-responsive to treatment with an antiepileptic drug (AED). Thesubject may eat a ketogenic diet. The epilepsy disorder may be anepilepsy disorder in an adult (e.g. more than about 16 years old).

The epilepsy disorder may be an epilepsy disorder in children. Thus, theepilepsy disorder may be a pediatric epilepsy disorder. The child may beless than about 1 week old. The child may be less than about 1 monthold. The child may be less than about 6 months old. The child may beless than about 12 months old. The child may be less than about 2 yearsold. The child may be less than about 3 years old. The child may be lessthan about 4 years old. The child may be less than about 5 years old.The child may be less than about 6 years old. The child may be less thanabout 7 years old. The child may be less than about 8 years old. Thechild may be less than about 9 years old. The child may be less thanabout 10 years old. The child may be less than about 12 years old.

The child may be more than about 1 week old. The child may be more thanabout 1 month old. The child may be more than about 6 months old. Thechild may be more than about 12 months old. The child may be more thanabout 2 years old. The child may be more than about 3 years old. Thechild may be more than about 4 years old. The child may be more thanabout 5 years old. The child may be more than about 5 years old. Thechild may be more than about 6 years old. The child may be more thanabout 7 years old. The child may be more than about 8 years old. Thechild may be more than about 9 years old. The child may be more thanabout 10 years old. The child may be more than about 11 years old. Thechild may be more than about 12 years old.

The child may have an epilepsy disorder treated by administering an AEDas described herein. Thus, the 5-HT receptor agonist or apharmaceutically acceptable salt thereof may be administered to such achild (e.g. as an add-on therapy).

In another aspect, is a method of treating Dravet Syndrome. The methodof treating Dravet Syndrome includes administering to a subject in needthereof, a therapeutically effective amount of the 5-HT receptor agonistor a pharmaceutically acceptable salt thereof. The method of treatingDravet Syndrome may include administering to a subject in need thereof,a pharmaceutical composition of the 5-HT receptor agonist or apharmaceutically acceptable salt thereof as described herein. The 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)may be co-administered to a subject in need thereof with an AED asdescribed herein.

In the methods described herein, the 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) may be co-administered withan anti-epileptic drug (AED). The AED may be acetazolamide,benzodiazepine, cannabadiols, carbamazepine, clobazam, clonazepam,eslicarbazepine acetate, ethosuximide, ethotoin, felbamate,fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A,lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine,perampanel, piracetam, phenobarbital, phenytoin, potassium bromide,pregabalin, primidone, retigabine, rufinamide, valproic acid, sodiumvalproate, stiripentol, tiagabine, topiramate, vigabatrin, orzonisamide. The AED may be valproic acid, sodium valproate, clonazepam,ethosuximide, felbamate, gabapentin, carbamazepine, oxcarbazepine,lamotrigine, levetiracetam, benzodiazepine, phenobarbital, pregabalin,primidone, tiagabine, topiramate, potassium bromide, phenytoin,stiripentol, vigabatrin, or zonisamide. The AED may be valproic acid,sodium valproate, gabapentin, topiramate, carbamazepine, oxcarbazepine,or vigabatrin.

The AED may be acetazolamide. The AED may be benzodiazepine. The AED maybe cannabadiols. The AED may be carbamazepine. The AED may be clobazam.The AED may be clonazepam. The AED may be eslicarbazepine acetate. TheAED may be ethosuximide. The AED may be ethotoin. The AED may befelbamate. The AED may be fenfluramine. The AED may be fosphenytoin. TheAED may be gabapentin. The AED may be ganaxolone. The AED may behuperzine A. The AED may be lacosamide. The AED may be lamotrigine. TheAED may be levetiracetam. The AED may be nitrazepam. The AED may beoxcarbazepine. The AED may be perampanel. The AED may be piracetam. TheAED may be phenobarbital. The AED may be phenytoin. The AED may bepotassium bromide. The AED may be pregabalin. The AED may be primidone.The AED may be retigabine. The AED may be rufinamide. The AED may bevalproic acid. The AED may be sodium valproate. The AED may bestiripentol. The AED may be tiagabine. The AED may be topiramate. TheAED may be vigabatrin. The AED may be zonisamide. Clemizole or aclemizole analog (including pharmaceutically acceptable salts thereof)or a pharmaceutical composition of clemizole or a clemizole analog maybe administered as an adjunctive therapy to one or more of the AEDsdescribed herein.

The 5-HT receptor agonist or a pharmaceutically acceptable salt thereofmay thus be administered as an add-on (e.g. in combination with) AEDmedications for treating seizures, including seizures associated withthe epilepsy disorders described herein. The 5-HT receptor agonist or apharmaceutically acceptable salt thereof may be administered as anadjunctive therapy (e.g. in combination with) AED medications fortreating seizures, including seizures associated with the epilepsydisorders described herein.

The epilepsy disorder may be characterized by partial seizures orgeneralized seizures. The epilepsy disorder may be characterized bypartial seizures. The epilepsy disorder may be characterized bygeneralized seizures. The partial seizure may be a simple focal seizure,a complex focal seizure, or a partial focal seizure with secondarygeneralization. The generalized seizure may be a generalizedtonic-clonic seizure, an absence seizure (i.e. petit mal), a myoclonicseizure, a clonic seizure, a tonic seizure, or a atonic seizure.

When co-administered with the AEDs described herein, the 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) and theAED may be administered simultaneously. When administeredsimultaneously, the 5-HT receptor agonist (including pharmaceuticallyacceptable salts thereof) may be formulated together with the AED (i.e.in a single dosage unit). The 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) may be formulated forseparate administration from the AED but administered at the same time.When co-administered with AEDs described herein, the 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) may beadministered sequentially (e.g. before or after) the administration ofthe AED. As set forth herein, one skilled in the art could readilydetermine the sequential order of administration.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered at a dose of about 1 mg/kg to about 1000mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptablesalts thereof) may be administered at a dose of about 10 mg/kg to about1000 mg/kg. The 5-HT receptor agonist (including pharmaceuticallyacceptable salts thereof) may be administered at a dose of about 10mg/kg to about 600 mg/kg. The 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) may be administered at a doseof about 25 mg/kg to about 500 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 25 mg/kg to about 400 mg/kg. The 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)may be administered at a dose of about 25 mg/kg to about 350 mg/kg. The5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered at a dose of about 25 mg/kg to about 300mg/kg. The 5-HT receptor agonist (including pharmaceutically acceptablesalts thereof) may be administered at a dose of about 25 mg/kg to about250 mg/kg. The 5-HT receptor agonist (including pharmaceuticallyacceptable salts thereof) may be administered at a dose of about 25mg/kg to about 200 mg/kg. The 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) may be administered at a doseof about 25 mg/kg to about 150 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 25 mg/kg to about 100 mg/kg. The 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)may be administered at a dose of about 25 mg/kg to about 75 mg/kg. The5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered at a dose of about 25 mg/kg to about 50mg/kg. As used herein “mg/kg” refers to mg per kg body weight of thesubject. Dosages described herein include administration of the 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)as a single therapeutic active agent or administration of the 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)as a therapeutic active agent in combination (as described herein) withan AED described herein.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered at a dose of about 1 mg/kg. The 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)may be administered at a dose of about 5 mg/kg. The 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 10 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 20 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 25 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 30 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 40 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 50 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 75 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 100 mg/kg.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered at a dose of about 125 mg/kg. The 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)may be administered at a dose of about 150 mg/kg. The 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 175 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 200 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 225 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 250 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 275 mg/kg.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered at a dose of about 300 mg/kg. The 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)may be administered at a dose of about 325 mg/kg. The 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 350 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 375 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 400 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 425 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 450 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 475 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 500 mg/kg.

The 5-HT receptor agonist analog (including pharmaceutically acceptablesalts thereof) may be administered at a dose of about 600 mg/kg. The5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered at a dose of about 700 mg/kg. The 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)may be administered at a dose of about 800 mg/kg. The 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 900 mg/kg. The 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) may beadministered at a dose of about 1000 mg/kg.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered in the dosages described herein at leastonce a day (e.g. once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12hours). The 5-HT receptor agonist (including pharmaceutically acceptablesalts thereof) may be administered daily in the dosages describedherein. The 5-HT receptor agonist (including pharmaceutically acceptablesalts thereof) may be administered at least twice a week in the dosagesdescribed herein. The 5-HT receptor agonist (including pharmaceuticallyacceptable salts thereof) may be administered at least three times aweek as described herein. The 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) may be administered monthlyas described herein.

Provided herein are methods of treating a disease or disorder caused bya deficiency of serotonin in the brain or under activity of one or more5HT receptors by administering a therapeutically effective amount ofclemizole, a clemizole analog, or a pharmaceutically acceptable saltthereof. The disease or disorder caused by a deficiency of serotonin inthe brain or under activity of one or more 5HT receptors may be amigraine. The disease or disorder caused by a deficiency of serotonin inthe brain or under activity of one or more 5HT receptors may be FragileX syndrome. The disease or disorder caused by a deficiency of serotoninin the brain or under activity of one or more 5HT receptors may bePrader-Willi syndrome. The disease or disorder caused by a deficiency ofserotonin in the brain or under activity of one or more 5HT receptorsmay be schizophrenia. The disease or disorder caused by a deficiency ofserotonin in the brain or under activity of one or more 5HT receptorsmay be depression. The disease or disorder caused by a deficiency ofserotonin in the brain or under activity of one or more 5HT receptorsmay be Alzheimer's disease. The disease or disorder caused by adeficiency of serotonin in the brain or under activity of one or more5HT receptors may be autism. The disease or disorder caused by adeficiency of serotonin in the brain or under activity of one or more5HT receptors may be neuropathic pain. The disease or disorder caused bya deficiency of serotonin in the brain or under activity of one or more5HT receptors may be Parkinson's disease. The disease or disorder causedby a deficiency of serotonin in the brain or under activity of one ormore 5HT receptors may be irritable bowel disorder. The disease ordisorder caused by a deficiency of serotonin in the brain or underactivity of one or more 5HT receptors may be dementia.

Further provided herein is a method of modulating activity of a 5HTreceptor comprising contacting a 5HT receptor with clemizole, aclemizole analog, or a pharmaceutically acceptable salt thereof.

The analog of clemizole may include compounds of formula (I) describedherein and may include compounds of similar structure as set forth, infor example, PCT/US2008/076804, WO10107739, WO2009039248, or U.S. Pat.No. 4,011,322, which are incorporated herein by reference.

In embodiments, the disclosure provides methods of treating epilepsy orDravet syndrome by administering a therapeutically effective amount oftrazadone or a pharmaceutically acceptable salt thereof. In embodiments,the therapeutically effective amount is from about 10 mg/day to about600 mg/day. In embodiments, the methods are for treating epilepsy. Inembodiments, the epilepsy is pediatric epilepsy. In embodiments, themethods are for treating Dravet syndrome.

Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising a 5-HTreceptor agonist or a pharmaceutically acceptable salt thereof usefulfor treating the aforementioned diseases and disorders. Thepharmaceutical composition may be formulated as a tablet, a powder, acapsule, a pill, a cachet, or a lozenge as described herein. Thepharmaceutical composition may be formulated as a tablet, capsule, pill,cachet, or lozenge for oral administration. The pharmaceuticalcomposition may be formulated for dissolution into a solution foradministration by such techniques as, for example, intravenousadministration. The pharmaceutical composition may be formulated fororal administration, suppository administration, topical administration,intravenous administration, intraperitoneal administration,intramuscular administration, intralesional administration, intrathecaladministration, intranasal administration, subcutaneous administration,implantation, transdermal administration, or transmucosal administrationas described herein.

When administered as pharmaceutical composition, the pharmaceuticalcompositions may include optical isomers, diastereomers, enantiomers,isoforms, polymorphs, hydrates, solvates or products, orpharmaceutically acceptable salts of the 5-HT receptor agonist. The 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)included in the pharmaceutical composition may be covalently attached toa carrier moiety, as described above. Alternatively, the 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) includedin the pharmaceutical composition is not covalently linked to a carriermoiety.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) may be administered alone or co-administered to a subject inneed thereof with an AED as described herein. Co-administration is meantto include simultaneous or sequential administration as described hereinof the 5-HT receptor agonist individually or in combination (e.g. morethan one compound—e.g. an AED described herein). The preparations canalso be combined, when desired, with other active substances (e.g. toprevent seizures).

Formulations

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) or a pharmaceutical composition described herein can beprepared and administered in a wide variety of oral, parenteral, andtopical dosage forms. Thus, the 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) or a pharmaceuticalcomposition described herein can be administered by injection (e.g.intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, or intraperitoneally). Also, the 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) or apharmaceutical composition described herein can be administered byinhalation, for example, intranasally. Additionally, the 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) or apharmaceutical composition can be administered transdermally. It is alsoenvisioned that multiple routes of administration (e.g., intramuscular,oral, transdermal) can be used to administer the 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) or apharmaceutical composition comprising same. The pharmaceuticalcompositions described herein may include a pharmaceutically acceptablecarrier or excipient and one or more of a 5-HT receptor agonist(including pharmaceutically acceptable salts thereof). Thepharmaceutical compositions described herein may include apharmaceutically acceptable carrier or excipient, one or more of a 5-HTreceptor agonist (including pharmaceutically acceptable salts thereof)and an one or more AED as described herein.

Preparation may include pharmaceutically acceptable carriers. Thepharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier may beone or more substance that may also act as diluents, flavoring agents,binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier may be a finely divided solid in a mixture withthe finely divided active component. In tablets, the active componentmay be mixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from 5% to 70% of the activecompound. Suitable carriers are magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. The term “preparation” is intended to include theformulation of the active compound with encapsulating material as acarrier providing a capsule in which the active component with orwithout other carriers, is surrounded by a carrier, which is thus inassociation with it. Similarly, cachets and lozenges are included.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; talc; pectin; dextrin; starch;tragacanth; a low melting wax; cocoa butter; carbohydrates; sugarsincluding, but not limited to, lactose, sucrose, mannitol, or sorbitol,starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins including, but not limited to, gelatin and collagen. Ifdesired, disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofthe 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) or a pharmaceutical composition (i.e., dosage). Pharmaceuticalpreparations described herein can also be used orally using, forexample, push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a coating such as glycerol or sorbitol.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

When parenteral application is needed or desired, particularly suitableadmixtures for the 5-HT receptor agonist (including pharmaceuticallyacceptable salts thereof) or a pharmaceutical composition comprisingsame are injectable, sterile solutions, preferably oily or aqueoussolutions, as well as suspensions, emulsions, or implants, includingsuppositories. In particular, carriers for parenteral administrationinclude aqueous solutions of dextrose, saline, pure water, ethanol,glycerol, propylene glycol, peanut oil, sesame oil,polyoxyethylene-block polymers, and the like. Ampoules are convenientunit dosages. The 5-HT receptor agonist (including pharmaceuticallyacceptable salts thereof) or a pharmaceutical composition comprisingsame can also be incorporated into liposomes or administered viatransdermal pumps or patches. Pharmaceutical admixtures suitable for useherein include those described, for example, in Pharmaceutical Sciences(17th Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309, the teachings ofboth of which are hereby incorporated by reference.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Also included herein are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can contain a thickening agent, such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents can be added to provide apalatable oral preparation, such as glycerol, sorbitol or sucrose. Theseformulations can be preserved by the addition of an antioxidant such asascorbic acid. As an example of an injectable oil vehicle, see Minto, J.Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical formulationsdescribed herein can also be in the form of oil-in-water emulsions. Theoily phase can be a vegetable oil or a mineral oil, described above, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg according to the particularapplication and the potency of the active component. The compositioncan, if desired, also contain other compatible therapeutic agents.

Formulations may include a surfactant or other appropriate co-solvent inthe composition. Such co-solvents include: Polysorbate 20, 60, and 80;Pluronic F-68, F-84, and P-103; cyclodextrin; and polyoxyl 35 castoroil. Such co-solvents are typically employed at a level between about0.01% and about 2% by weight. Viscosity greater than that of simpleaqueous solutions may be desirable to decrease variability in dispensingthe formulations, to decrease physical separation of components of asuspension or emulsion of formulation, and/or otherwise to improve theformulation. Such viscosity-building agents include, for example,polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxy propyl cellulose, chondroitin sulfate and salts thereof,hyaluronic acid and salts thereof, and combinations of the foregoing.Such agents are typically employed at a level between about 0.01% andabout 2% by weight.

Viscosity greater than that of simple aqueous solutions may be desirableto decrease variability in dispensing the formulations, to decreasephysical separation of components of a suspension or emulsion offormulation and/or otherwise to improve the formulation. Such viscositybuilding agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose, chondroitin sulfate and salts thereof, hyaluronic acid andsalts thereof, combinations of the foregoing, and other agents known tothose skilled in the art. Such agents are typically employed at a levelbetween about 0.01% and about 2% by weight. Determination of acceptableamounts of any of the above adjuvants is readily ascertained by oneskilled in the art.

The pharmaceutical compositions may additionally include components toprovide sustained release and/or comfort. Such components include highmolecular weight, anionic mucomimetic polymers, gelling polysaccharides,and finely-divided drug carrier substrates. These components arediscussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841;5,212,162; and 4,861,760. The entire contents of these patents areincorporated herein by reference in their entirety for all purposes.

The pharmaceutical composition may be intended for intravenous use. Thepharmaceutically acceptable excipient can include buffers to adjust thepH to a desirable range for intravenous use. Many buffers includingsalts of inorganic acids such as phosphate, borate, and sulfate areknown.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) or a pharmaceutical composition thereof can be deliveredtransdermally, for treating the epilepsy disorders described herein, bya topical route, formulated as applicator sticks, solutions,suspensions, emulsions, gels, creams, ointments, pastes, jellies,paints, powders, and aerosols.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) can be provided as a salt in the pharmaceutical compositionsdescribed herein and can be formed with many acids, including but notlimited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic,succinic, etc. Salts tend to be more soluble in aqueous or otherprotonic solvents that are the corresponding free base forms.

The 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) or a pharmaceutical composition thereof administered fortreating epilepsy disorders described herein may be administered viaparenteral administration, such as intravenous (IV) administration oradministration into a body cavity or lumen of an organ. The formulationsfor administration will commonly comprise a solution of the compositionsof the present invention dissolved in a pharmaceutically acceptablecarrier. Among the acceptable vehicles and solvents that can be employedare water and Ringer's solution, an isotonic sodium chloride. Inaddition, sterile fixed oils can conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of the compositions of the present invention inthese formulations can vary widely, and will be selected primarily basedon fluid volumes, viscosities, body weight, and the like, in accordancewith the particular mode of administration selected and the patient'sneeds. For IV administration, the formulation can be a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension can be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation can also be asterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

The pharmaceutical formulations of the 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) for treating an epilepsydisorder can be delivered by the use of liposomes which fuse with thecellular membrane or are endocytosed, i.e., by employing ligandsattached to the liposome, or attached directly to the oligonucleotide,that bind to surface membrane protein receptors of the cell resulting inendocytosis. By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the compositions of the present invention into the target cells invivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996;Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp.Pharm. 46:1576-1587, 1989).

Co-administration includes administering one active agent (e.g.clemizole or a clemizole analog (including pharmaceutically acceptablesalts thereof)) within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hoursof a second active agent (e.g. an anticonvulsant). Co-administration mayinclude administering one active agent within 0.5, 1, 2, 4, 6, 8, 10,12, 16, 20, or 24 hours of a second active agent. Co-administration mayinclude administering two active agents simultaneously, approximatelysimultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes ofeach other), or sequentially in any order. Co-administration can beaccomplished by co-formulation, i.e., preparing a single pharmaceuticalcomposition including both active agents. In other embodiments, theactive agents can be formulated separately. The active and/or adjunctiveagents may be linked or conjugated to one another.

Co-administration also includes combination with treatments for epilepsydisorders such as dietary requirements or dietary changes. Accordingly,the 5-HT receptor agonist (including pharmaceutically acceptable saltsthereof) or a pharmaceutical composition thereof may be administered tosubjects on specialized diets, including but not limited to, a ketogenicdiet (e.g. a high-fat, adequate-protein, low-carbohydrate diet).

Effective Dosages

The pharmaceutical composition may include the 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. For example, when administered in methods to treat an epilepsydisorder (e.g. Dravet Syndrome), such compositions will contain amountsof the 5-HT receptor agonist (including pharmaceutically acceptablesalts thereof) or a pharmaceutical composition thereof effective toachieve the desired result (e.g. inhibiting seizures).

The dosage and frequency (single or multiple doses) of the 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) or apharmaceutical composition thereof administered can vary depending upona variety of factors, including route of administration; size, age, sex,health, body weight, body mass index, and diet of the recipient; natureand extent of symptoms of the disease being treated; presence of otherdiseases or other health-related problems; kind of concurrent treatment;and complications from any disease or treatment regimen. Othertherapeutic regimens or agents can be used in conjunction with themethods described herein.

The therapeutically effective amounts of the 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) or apharmaceutical composition thereof for treating epilepsy diseasesdescribed herein may be initially determined from cell culture assays.Target concentrations will be those concentrations of the 5-HT receptoragonist (including pharmaceutically acceptable salts thereof) or apharmaceutical composition thereof capable of inhibiting or otherwisedecreasing seizures experienced by a patient.

Therapeutically effective amounts of the 5-HT receptor agonist(including pharmaceutically acceptable salts thereof) or apharmaceutical composition thereof for use in humans may be determinedfrom animal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring response of thepatient to the treatment and adjusting the dosage upwards or downwards,as described above.

Dosages may be varied depending upon the requirements of the subject andthe compound being employed. The dose administered to a subject, in thecontext of the pharmaceutical compositions presented herein, should besufficient to effect a beneficial therapeutic response in the subjectover time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side effects. Generally,treatment is initiated with smaller dosages, which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered the 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) or a pharmaceuticalcomposition thereof effective for the particular epilepsy disorder beingtreated. This will provide a therapeutic regimen that is commensuratewith the severity of the individual's disease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of the 5-HT receptor agonist (includingpharmaceutically acceptable salts thereof) or a pharmaceuticalcomposition thereof by considering factors such as potency, relativebioavailability, patient body weight, presence and severity of adverseside effects, preferred mode of administration, and the toxicity profileof the selected agent.

Toxicity

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975.The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

When parenteral application is needed or desired, particularly suitableadmixtures for the 5-HT receptor agonist (including pharmaceuticallyacceptable salts thereof) included in the pharmaceutical composition maybe injectable, sterile solutions, oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-block polymers, and thelike. Ampoules are convenient unit dosages. Pharmaceutical admixturessuitable for use in the pharmaceutical compositions presented herein mayinclude those described, for example, in Pharmaceutical Sciences (17thEd., Mack Pub. Co., Easton, Pa.) and WO 96/05309, the teachings of bothof which are hereby incorporated by reference.

EXAMPLES Example 1

Epilepsy can be acquired as a result of an injury to the brain orgenetic mutation. Among the genetic epilepsies more than 650 variantshave been identified in the SCN1A gene (Harkin, L. A. et al. Thespectrum of SCN1A-related infantile epileptic encephalopathies. Brain130, 843-852 (2007); Mulley J. C., et al., SCN1A mutations and epilepsy.Hum. Mutat. 25, 535-542 (2005)). Missense or frame-shift mutations inthis gene are associated with generalized epilepsy with febrile seizuresplus (GEFS+) (Ceulemans, B. P., et al., Clinical correlations ofmutations in the SCN1A gene: from febrile seizures to severe myoclonicepilepsy in infancy. Pediatric Neurol. 30, 236-243 (2004)) as well as amore severe disorder known as Dravet syndrome. Children with DSinitially exhibit normal development but often experience febrileseizure episodes within the first year of life with eventual progressionto severe spontaneous recurrent seizures, intellectual disability,ataxia, and psychomotor dysfunction. Seizures are inadequately managedusing available antiepileptic drugs (AEDs) and these children are poorcandidates for neurosurgical resection (Bender, A. C., et al., SCN1Amutations in Dravet syndrome: Impact of interneuron dysfunction onneural networks and cognitive outcome. Epilepsy Beh. 23, 177-186(2012)).

In mammalian brain there are four main subtypes of voltage-gated sodiumchannel alpha subunits: Na_(v)1.1, Na_(v)1.2, Na_(v)1.3 and Na_(v)1.6,encoded for by the genes SCN1A, SCN2A, SCN3A, and SCN8A, respectively.Opening of these channels produces a sodium conductance and rapid cellmembrane depolarization e.g., features integral to action potentialinitiation (Catterall, W. A., et al., Na_(v)1.1 channels and epilepsy.J. Physiol. 588, 1849-1859 (2010)). In mice, Na_(v)1.1 is widelyexpressed in the central nervous system including the axon initialsegment of parvalbumin-positive hippocampal interneurons and excitatoryprincipal cells (Kim, D. Y., et al., Reduced sodium channel Na(v)1.1levels in BACE1-null mice. J. Biol. Chem. 286, 8106-8116 (2011); Chen,C., et al., Mice lacking sodium channel betal subunits display defectsin neuronal excitability, sodium channel expression, and nodalarchitecture. J. Neurosci. 24, 4030-4042 (2004)). Heterozygous deletionof Na_(v)1.1 in mice leads to a reduction in the firing capability ofacutely dissociated fast-spiking interneurons (Yu, F. H., et al.,Reduced sodium current in GABAergic interneurons in a mouse model ofsevere myoclonic epilepsy in infancy. Nat. Neurosci. 9, 1142-1149(2006)). Mice with global or interneuron-specific heterozygous deletionof Na_(v)1.1 exhibit temperature-induced and spontaneous seizures, mildataxia, autism-like behaviors and premature death (Yu, F. H., et al.,Reduced sodium current in GABAergic interneurons in a mouse model ofsevere myoclonic epilepsy in infancy. Nat. Neurosci. 9, 1142-1149(2006); Oakley, J. C., et al., Temperature- and age-dependent seizuresin a mouse model of severe myoclonic epilepsy in infancy. Proc. Natl.Acad. Sci. USA 106, 3994-3999 (2009); Cheah, C. S., et al., Specificdeletion of Na_(v)1.1 sodium channels in inhibitory interneurons causesseizures and premature death in a mouse model of Dravet syndrome. Proc.Natl. Acad. Sci. USA 109, 14646-14651 (2012)). Knock-in mouse carrying apremature stop codon in domain III of the Na_(v)1.1 channel also exhibita decrement in spike amplitude during prolonged interneuron firing andincreased sensitivity to temperature-induced seizures (Ogiwara, I., etal., Na_(v)1.1 localizes to axons of parvalbumin-positive inhibitoryinterneurons: a circuit basis for epileptic seizures in mice carrying anScn1a gene mutation. J. Neurosci. 27, 5903-5914 (2007)).

Generation and characterization of valid animal models is critical toefforts to understand the pathophysiology of DS, and to aid inidentification of novel therapies. While considerable attention hasfocused on modeling SCN1A mutations in mice these animals have provendifficult to breed and epilepsy phenotypes are strongly influenced bybackground strain genetics. Induced pluripotent stem cells can begenerated from DS patients but individual neurons do not recapitulatethe network environment necessary for in vivo seizure generation. Daniorerio (zebrafish), a simple vertebrate species, provide an alternativemodel system with significant advantages for genetic manipulation,cost-efficient breeding and in vivo drug discovery (Lessman, C. A., Thedeveloping zebrafish (Danio rerio): a vertebrate model forhigh-throughput screening of chemical libraries. Birth Defects Res. C.Embryo Today 93, 268-280 (2011); Delvecchio, C., et al., The zebrafish:a powerful platform for in vivo, HTS drug discovery. Assay Drug Dev.Technol. 9, 354-361 (2011); Rinkwitz, S., et al., Zebrafish: anintegrative system for neurogenomics and neurosciences. Prog. Neurobiol.93, 231-243 (2011)). Ideally, an animal model should be based on a knowngenetic cause of the disease (SCN1A mutation), accurately recapitulatekey features of the disease (epilepsy), and respond, or not, totherapies commonly used in patients with the disease (pharmacologicalvalidation). If successful, such a model could inform the understandingof the disease process and catalyze explorations toward new therapies.

In zebrafish, the voltage-gated sodium channel family consists of foursets of duplicated genes: scn1Laa & scn1Lab, scn4aa & scn4ab, scn5Laa &scn5Lab, and scn8aa & scn8ab (Novak, A. E., et al., Embryonic and larvalexpression of zebrafish voltage-gated sodium channel alpha-subunitgenes. Dev. Dyn. 235, 1962-1973 (2006)). The zebrafish scn1Lab geneshares a 77% identity with human SCN1A and is expressed in the centralnervous system. A homozygous zebrafish mutant for this gene (originallytermed didy^(s552)) was discovered in a chemical mutagenesis screenusing the optokinetic response as an assay (Schoonheim, P. J.,Arrenberg, A. B., Del Bene, F., & Baier H., Optogenetic localization andgenetic perturbation of saccade-generating neurons in zebrafish. J.Neurosci. 30, 7111-7120 (2010)). These types of screens are based oninducing random point mutations using the alkylating agentN-ethyl-N-nitrosourea (ENU), resulting mutations are typicallyloss-of-function and recessive. Although this is a homozygous mutation,scn1Lab zebrafish mutants are relevant for the autosomal dominant humanDravet Syndrome given the genome duplication in zebrafish and thepresence of an additional Na_(v)1.1 homologue (scn1Laa). scn1Lab mutantswere characterized at the molecular and behavioral level, demonstratedthat mutants exhibit spontaneous drug-resistant seizures, and then usedthem in a novel high-throughput screening program to identify compoundsthat ameliorate the epilepsy phenotype. A phenotype-based screenidentified clemizole, an FDA-approved compound, as an effectiveinhibitor of spontaneous convulsive behaviors and electrographicseizures in these mutants.

scn1Lab expression and characterization of mutant zebrafish. Zebrafishwith a mutation in domain III of a voltage-gated sodium channel wereidentified by Dr. Herwig Baier during a chemical mutagenesis screen(Schoonheim, P. J., Arrenberg, A. B., Del Bene, F., & Baier H.,Optogenetic localization and genetic perturbation of saccade-generatingneurons in zebrafish. J. Neurosci. 30, 7111-7120 (2010)). Originalscn1Lab mutants were backcrossed onto the Tupfel long (TL) backgroundfor 7-10 generations and confirmed a methionine (M) to arginine (R)mutation in the colony (FIG. 1A). Reverse transcriptase (RT) andquantitative (q) PCR revealed a decrease in mRNA expression for scn1Labin mutant larvae at 3, 5 and 7 days post-fertilization (dpf) (FIG. 1B);antibodies recognizing this protein in zebrafish are not available. Asexpected (Novak, A. E., et al., Embryonic and larval expression ofzebrafish voltage-gated sodium channel alpha-subunit genes. Dev. Dyn.235, 1962-1973 (2006)), scn1Lab is prominently expressed during earlystages of larval development (FIG. 1B) and specifically in the centralnervous system at 3 dpf (FIGS. 1D, E). Whole-mount in situ hybridizationrevealed diffuse but prominent expression in brain regions correspondingto forebrain (telencephalon), optic tectum and cerebellum. A similarexpression pattern was observed for scn1Laa at 3 dpf. At 5 and 7 dpf,CNS expression remained prominent and faint scn1Lab signal was alsonoted in the heart (FIG. 1D). Relative expression of scn8aa or scn8ab(Na_(v)1.6) e.g., a subunit thought to act as a genetic modifier of DS(Martin, M. S., et al., The voltage-gated sodium channel Scn8a is agenetic modifier of severe myoclonic epilepsy of infancy. Hum. Mol. Gen.16, 2892-2899 (2007)), failed to reveal a significant difference inexpression between mutants and sibling controls at 5 dpf (FIG. 1C).Similarly, microarray analysis at 5 dpf also failed to detect acompensatory change in the mRNA expression of thirteen differentzebrafish scn subunits (Table I) including the other homolog (scn1Laa).These results demonstrate a selective defect in a zebrafish Na_(v)1.1gene expressed in the CNS during early development.

Large-scale transcriptomic analysis of scn1Lab mutants. Althoughinherited disorders of voltage-gated ion channels are recognized as anetiology of epilepsy, investigation of transcriptional changes has notbeen reported for any epilepsy-related channelopathy. To detectdifferences in gene expression in an unbiased manner an Agilent Daniorerio chip covering 44,000 probes (FIGS. 2A, B) was used. Hierarchicalclustering analyses showed that ˜2.5% (1099) of these probes weredifferentially expressed between mutants and sibling controls at 5 dpf(p≦0.01, t test; 674 up-regulated and 425 down-regulated); 405 wereassigned to an “unknown function” category. A list of 30 down- andup-regulated known genes showing the greatest differences in expressionis shown in FIG. 2C. These differences were modest as 90% (990/1099) ofthe identified genes exhibited fold-changes between 0.8 and 2.0. Similarto microarray analysis of Mecp2 single-gene mutant mice (Jordan, C., etal., Cerebellar gene expression profiles of mouse models for Rettsyndrome reveal novel MeCP2 targets. BMC Med. Genet. 8, 36 (2007)), manyof the genes identified had no obvious CNS-related function and/orexpression.

The two largest fold-changed genes, somatolactin β and a Na, K-ATPase,have expression primarily restricted to the pituitary (smt1b) (Lopez,M., et al., Expression of the somatolactin β gene during zebrafishembryonic development. Gene Expr. Patterns 6, 156-161 (2006)) or ear,intestinal bulb and pronephric duct (atp1a1a.5) (Blasiole, B., et al.,Cloning, mapping, and developmental expression of a sixth zebrafish Na,K-ATPase alpha1 subunit gene (atp1a1a.5). Mech. Dev. 119, Suppl1:S211-S214 (2002)). Probes for several genes related to apoptosis(casp8, casp8b and casp3b) did not reveal any statistically significantchanges in the microarray studies. Of the genes with altered expressionin scn1Lab mutants, six were previously implicated in neurologicaldisorders e.g., pcdh19 (infantile epileptic encephalopathy), cyfip1 andfxr2 (Fragile X syndrome), ocr1 (Lowe syndrome), ubap21 (Parkinson'sdisease) and oca2 (Angelman syndrome). Microarray-based gene expressionmeasurements were verified for 14 randomly selected genes using qPCR(FIG. 3A).

Biological functions were assigned to all genes using gene ontology (GO)annotations and the 482 genes showing at least a 1.5-fold change inexpression and a p value <0.01 were categorized further (FIG. 3C).Calcium ion binding genes include annexin A1c, A1b and 2a, spectrin α2,neurexin 2a, calsyntenin 1 and parvalbumin 3. Significant changes in agap junction channel (cx43), a gene involved in clustering ofvoltage-gated sodium channels at the axon initial segment (spna2) andthe ubiquitin domain of a GABA receptor (map11c3b) were also noted.Three additional genes not found on the microarray were chosen for qPCRanalysis (FIG. 3B): hcn1, a gene shown to be correlated with SCN1A usingdata mining and down-regulated in several seizure models (Noam, Y., etal., Towards an integrated view of HCN channel role in epilepsy. Curr.Opin. Neurobiol. 21, 873-879 (2011)) was significantly reduced inscn1Lab mutants compared to sibling control (p<0.05 2-tail Student'st-test). However, homer and bdnf e.g., genes involved in synaptogenesisrelated to the formation of recurrent excitatory synapses and epilepsy(Avedissian, M., et al., Hippocampal gene expression analysis using theORESTES methodology shows that homer 1a mRNA is upregulated in the acuteperiod of the pilocarpine epilepsy model. Hippocampus 17, 130-136(2007); Tongiorgi, E., et al., Brain-derived neurotrophic factor mRNAand protein are targeted to discrete dendritic laminas by events thattrigger epileptogenesis. J. Neurosci. 24, 6842-6852 (2004)) wereunchanged.

Spontaneous seizures in scn1Lab mutant zebrafish. scn1Lab mutants weremonitored for evidence of spontaneous electrographic seizures startingat 3 dpf e.g., the first larval stage at which epileptiform dischargecan be detected (Baraban, S. C., et al., A large-scale mutagenesisscreen to identify seizure-resistant zebrafish. Epilepsia 48, 1151-157(2007); Hortopan, G. A., et al., Spontaneous seizures and altered geneexpression in GABA signaling pathways in a mind bomb mutant zebrafish.J. Neurosci. 30, 13718-13728 (2010); Hunt, R. F., Hortopan, G. A.,Gillespie, A., & Baraban, S. C., A novel zebrafish model ofhyperthermia-induced seizures reveals a role for TRPV4 channels andNMDA-type glutamate receptors. Exp. Neurol. 237, 199-206 (2012);Baraban, S. C., Taylor, M. R., Castro, P. A., & Baier H.,Pentylenetetrazole induced changes in zebrafish behavior, neuralactivity and c-fos expression. Neuroscience 131, 759-768 (2005); Chege,S. W., Hortopan, G. A., Dinday, M. T., & Baraban, S. C., Expression andfunction of KCNQ channels in larval zebrafish. Dev. Neurobiol. 72,186-198 (2012)). Mutant larvae were identified by their “black”appearance (FIG. 4A), which is indicative of a defect in pigmentaggregation and die prematurely between 10 and 12 dpf, as reportedpreviously (Novak, A. E., et al., Embryonic and larval expression ofzebrafish voltage-gated sodium channel alpha-subunit genes. Dev. Dyn.235, 1962-1973 (2006)). Forebrain extracellular field recordings fromparalyzed and agar-immobilized scn1Lab mutants were marked by frequentbrief interictal-like bursts and large-amplitude long durationictal-like events starting at 3 dpf (n=4) and progressively becomingmore prominent between 4 and 7 dpf (n=132) (FIG. 2C). These events wereconfirmed in 100% of mutants at 3 dpf, 100% at 4 dpf, 97% at 5 dpf, 98%at 6 dpf and 100% at 7 dpf.

Abnormal electrical events were not observed in age-matched siblingcontrols at any developmental stage (n=36). Hyperthermia-inducedseizures (Hunt, R. F., Hortopan, G. A., Gillespie, A., & Baraban, S. C.,A novel zebrafish model of hyperthermia-induced seizures reveals a rolefor TRPV4 channels and NMDA-type glutamate receptors. Exp. Neurol. 237,199-206 (2012)) could be evoked in 5 dpf scn1Lab mutants and controls atapparently similar temperature thresholds (mutant: 26.9±0.5 C.°; n=14;control: 25.9±0.5 C.°; n=14; p=0.164 t-test). However, thesemeasurements were complicated, in mutants, by simultaneous occurrence ofhigh frequency spontaneous epileptiform discharges. Mutants had elevatedlevels of swim activity and exhibited unprovoked seizure-like behaviorconsisting of whole-body convulsions and rapid undirected movementstarting at 4 dpf (n=36). A representative locomotion tracking plot of ascn1Lab mutant showing hyperactivity and convulsive behavior is shown inFIG. 4B. This behavior is similar to that classified as a Stage IIIseizure in larvae exposed to pentylenetetrazole (Baraban, S. C., Taylor,M. R., Castro, P. A., & Baier H., Pentylenetetrazole induced changes inzebrafish behavior, neural activity and c-fos expression. Neuroscience131, 759-768 (2005)). Seizure behaviors were never observed in controlsat any stage of development (n=36). In pools of mutant and siblingcontrol larvae, scn1Lab mutants stay close to the sides of the petridish, which is considered a form of thigmotaxis in fish (Ellis, L. D.,Seibert, J., & Soanes, K. H., Distinct modes of induced hyperactivity inzebrafish larvae. Brain Res. 1449, 46-59 (2012)). These results reveal astriking epilepsy phenotype in scn1Lab mutant zebrafish.

Pharmacological evaluation of scn1Lab mutant zebrafish. Seizuresassociated with SCN1A mutations are poorly responsive to most AEDs. Toevaluate pharmaco-sensitivity spontaneous electrographic seizures wererecorded in agar-embedded scn1Lab mutants (5-6 dpf) under baselineconditions, and again after application of a commercially available AED.All drugs were bath applied at a concentration of 1 mM; seven fish weretested for each drug. Epileptiform event frequency (includinginterictal- and ictal-like discharges) and the fractional time spentseizing in scn1Lab mutants were reduced by valproate, diazepam,potassium bromide and stiripentol (FIGS. 5A, B, D). Burst durations werenot significantly changed for any of these drug exposures (FIG. 5C).

As expected, most AEDs had no effect and epileptiform activity becamemore frequent following exposure to carbamazepine (in 2 of 7 fish),ethosuximide (4 of 7 fish) or vigabatrin (6 of 7 fish). Because DSchildren often respond to the ketogenic diet (KD) (Dravet, C., et al.,Severe myoclonic epilepsy in infancy: Dravet syndrome. Adv. Neurol. 95,71-102 (2005)) a separate clutch of scn1Lab mutants was exposed,siblings and WT controls to a form of the diet for 48 hr starting at 4dpf. Locomotion tracking data on KD-exposed larvae at 6 dpf confirm areduction in seizure-like behavior to control levels in 7 of 10 mutants(Fig. E; mean velocity, treated mutants=0.43±0.09 mm/sec, n=16;un-treated mutants=0.81±0.05 mm/sec, n=28; p<0.05 Kruskal-Wallis ANOVAon Ranks with a Dunn's pairwise multiple comparison). No significantdifferences in swim behavior were noted in sibling controls treated withthe KD (mean velocity=0.63±0.05 mm/sec, n=20) compared to un-treated WTlarvae at 6 dpf (mean velocity=0.62±0.07 mm/sec; n=20). Acute exposure(20 min) to the diet had no effect on mutant seizure behavior in thelocomotion assay (n=14; change in mean velocity <34%). Subsequentforebrain field recordings obtained from the same zebrafish used in thelocomotion assay (FIG. 5F, top trace) confirmed the occurrence ofspontaneous epileptiform discharge for embryo media exposed scn1Labmutants and a suppression of burst activity in mutants exposed to the KDfor 48 hr (FIG. 5F, bottom trace). These results demonstrate that thepharmacological profile for scn1Lab mutants resembles that seen inchildren with DS.

High-throughput drug screening in scn1Lab mutants. Because behavioralseizure activity is easily and rapidly monitored using a locomotiontracking format (Baraban, S. C., et al., A large-scale mutagenesisscreen to identify seizure-resistant zebrafish. Epilepsia 48, 1151-157(2007); Hortopan, G. A., et al., Spontaneous seizures and altered geneexpression in GABA signaling pathways in a mind bomb mutant zebrafish.J. Neurosci. 30, 13718-13728 (2010); Baraban, S. C., Taylor, M. R.,Castro, P. A., & Baier H., Pentylenetetrazole induced changes inzebrafish behavior, neural activity and c-fos expression. Neuroscience131, 759-768 (2005); Chege, S. W., Hortopan, G. A., Dinday, M. T., &Baraban, S. C., Expression and function of KCNQ channels in larvalzebrafish. Dev. Neurobiol. 72, 186-198 (2012); Berghmans, S., Hunt, J.,Roach, A., & Goldsmith, P., Zebrafish offer the potential for a primaryscreen to identify a wide variety of potential anticonvulsants. EpilepsyRes. 75, 18-28 (2007); Baxendale, S., et al., Identification ofcompounds with anti-convulsant properties in a zebrafish model ofepileptic seizures. Dis. Model. Mech. 5, 773-774 (2012); Cario, C. L.,Farrell, T. C., Milanese, C., & Burton, E. A., Automated measurement ofzebrafish larval movement. J. Physiol. 589, 3703-3708 (2011); Winter, M.J., et al., Validation of a larval zebrafish locomotor assay forassessing the seizure liability of early-stage development drugs. J.Pharm. Tox. Methods 5, 176-187 (2008); Orellana-Paucar, A. M., et al.,Anticonvulsant activity of bisabolene sesquiterpenoids of Curcuma longain zebrafish and mouse seizure models. Epilepsy Beh. 24, 14-22 (2012)(FIGS. 4B and 5B1).

A high-throughput phenotype-based strategy was designed to screenchemical libraries for compounds that reduce mutant behavior to Stage 0(very little swim activity) or Stage I (increased, but non-convulsive,swim activity) e.g., behavior equivalent to that seen in normal WTmutants. Automated measurement of larval activity was achieved usingEthoVision tracking software (Noldus Information Technology) and ahigh-speed camera. Previous studies confirmed that high velocitymovement ≧20 mm/sec correspond to paroxysmal seizure-like convulsions(Stage III) (Winter, M. J., et al., Validation of a larval zebrafishlocomotor assay for assessing the seizure liability of early-stagedevelopment drugs. J. Pharm. Tox. Methods 5, 176-187 (2008);Orellana-Paucar, A. M., et al., Anticonvulsant activity of bisabolenesesquiterpenoids of Curcuma longa in zebrafish and mouse seizure models.Epilepsy Beh. 24, 14-22 (2012)).

Using a 96-well format, mutant swim activity at baseline wasautomatically tracked, and then again after addition of a test compound(100 μl); each compound was tested on 6 to 12 individual larvae at 5dpf. The change in mutant swim activity between two consecutiverecording epochs in embryo media was taken as baseline and is shown inFIG. 6A (n=28). Based on a standard deviation of 17.3 for baselinerecordings associated simply with a solution exchange, compounds thatinhibited movement (measured as a change in mean velocity) by ≧34% werescreened for. To validate this approach, eleven AEDs and the KD werefirst screened using this assay. As expected from electrophysiologicalassays (FIG. 5), diazepam, potassium bromide, stiripentol, valproate anda 48 hr exposure to KD effectively inhibited seizure behavior in thelocomotion-based assay (FIG. 6B); ganaxolone, a neuroactive steroidrelated to allopregnalone, was also effective. Next, test compounds werescreened at an initial concentration of 667 μM from a library thatincluded US Food and Drug Administration (FDA) approved and toxicologytested drugs.

Among the 320 compounds screened in vivo, 18 were found to significantlyinhibit spontaneous seizures in scn1Lab mutants to levels comparable toStage 0 or Stage I behavior and/or reduce mean swim velocity (redcircles in FIG. 6C). These 18 compounds were then re-tested on aseparate clutch of scn1Lab mutants at concentrations of 667, 67 and 6.7μM. In the initial screen, 81 compounds were identified as lethal i.e.,no visible heartbeat or movement in response to touch after a 30 minexposure and were re-evaluated at a dilution of 1:100; none of theseadvanced further. The drug library included a number of additionalcompounds with putative anticonvulsant properties (beclamide,aminohydroxybutyric acid, and tiletamine) that were also ineffective inthe 96-well locomotion assay at 667 μM. 14 of the re-tested compoundseither failed to successfully inhibit seizure behavior in a secondclutch of scn1Lab mutants or only suppressed behavior at the highestdrug concentration. Next 4 (out of 18) compounds that were effective inreducing seizure-induced swim activity and mean velocity at all threedrug concentrations for further testing were selected: zoxazolamine,clemizole HCl, clorgiline HCl and tolperisone HCl (FIG. 6D). Each ofthese compounds was evaluated a third time in the locomotion assay at aconcentration of 100 μM, and subsequently monitored for forebrainelectrographic activity. Clorgiline (a monoamine oxidase A inhibitor)and the muscle relaxants zoxazolamine (Hadra, R. & Millichap J. G.,Quantitative assessment of motor function in cerebral palsy: evaluationof zoxazolamine (flexin), a new muscular relaxant agent. Neurology 6,843-852 (1956)) and tolperisone (Sakitama, K., The effects of centrallyacting muscle relaxants on the intrathecal noradrenaline-inducedfacilitation of the flexor reflex mediated by group II afferent fibersin rats. Jpn. J. Pharmacol. 63, 369-736 (1993)) were identified as“false positives” because they reduced swim activity at thisconcentration but when the same mutant was embedded in agarelectrographic seizure events were still observed (see FIG. 6E).

Only one compound, clemizole (antihistamine and NS4B RNA bindinginhibitor) (Finkelstein, M., Kromer, C. M., Sweeney, S. A., & DelahuntC. S., Some aspects of the pharmacology of clemizole hydrochloride. J.Am. Pharm. Assoc. Am. Pharm. Assoc. 49, 18-22 (1960); Einav, S., Sobol,H. D., Gehrig, E., & Glenn J. S., Discovery of a hepatitis C target andits pharmacological inhibitors by microfluidic affinity analysis. Nat.Biotechnol. 26, 1019-1027 (2008)), was effective in suppressingspontaneous seizure activity in both assays (FIG. 6D-E). Clemizole hadno significant effect on seizure behavior in the locomotion assay atconcentrations between 6.25 and 50 μM (n=33). As an additionalevaluation of the therapeutic potential for acute clemizole treatment,it was demonstrated that 100 μM clemizole was effective in reducingseizure behavior in WT zebrafish exposed to 15 mM pentylenetetrazole(FIG. 6D; n=10) i.e., a model of acute seizures based on GABA receptorantagonism. These results suggest that scn1Lab mutants can be used in ahigh-throughput screen to identify potential lead compounds for Dravetsyndrome.

The scn1Lab zebrafish mutant described here is the first simplevertebrate model of a sodium channel mutation that recapitulatesfeatures of Dravet syndrome, a catastrophic form of drug-resistantepilepsy in children. These mutants exhibit hyperactivity, includingconvulsive behavior, spontaneous electrographic seizures, shortenedlifespan and a pharmacological profile similar to the human condition.Additional molecular analysis of scn1Lab mutants suggests the absence ofgross changes in global gene expression and a lack of compensation, atthe RNA level, by other voltage-gated Na⁺ channel subunits. A two-stagephenotype-based drug screening strategy to identify lead compounds withthe potential to ameliorate epilepsy phenotypes associated with SCN1Amutation identified one FDA-approved drug (clemizole).

Electroencephalographic (EEG) activity is typically normal in the firstyear of life for DS patients with an evolution to abnormal paroxysmaland polyspike activity between 1 and 9 years of age. This age-dependentpattern was mimicked in developing zebrafish larvae at ages where scn1aexpression was significant. Forebrain extracellular recordings in veryyoung larvae (3 dpf) appeared largely normal with the occasional smallburst of polyspike activity. Frequent brief interictal-like activitywith large amplitude polyspike burst discharges became more prominent aslarvae aged. The architecture of these electrical events resembled thosepreviously described in wild-type larvae exposed to pentylenetetrazole(Baraban, S. C., Taylor, M. R., Castro, P. A., & Baier H.,Pentylenetetrazole induced changes in zebrafish behavior, neuralactivity and c-fos expression. Neuroscience 131, 759-768 (2005)),4-aminopyridine (Baraban, S. C., et al., A large-scale mutagenesisscreen to identify seizure-resistant zebrafish. Epilepsia 48, 1151-157(2007)), linopirdine (Chege, S. W., Hortopan, G. A., Dinday, M. T., &Baraban, S. C., Expression and function of KCNQ channels in larvalzebrafish. Dev. Neurobiol. 72, 186-198 (2012)) or hyperthermia (Hunt, R.F., Hortopan, G. A., Gillespie, A., & Baraban, S. C., A novel zebrafishmodel of hyperthermia-induced seizures reveals a role for TRPV4 channelsand NMDA-type glutamate receptors. Exp. Neurol. 237, 199-206 (2012)).

The appearance of electrographic seizure activity corresponds withhyperactivity, full-body convulsions with associated high-velocity swimactivity and brief loss-of-posture in freely behaving mutants. Thesetypes of spontaneous behaviors are never observed in wild-type larvaeand, again, resemble those previously observed only during exposure toconvulsant drugs. These behaviors are an indirect indicator of seizureactivity and could be used for rapid in vivo evaluation of drugtreatments and lethality in a multi-well format using automatedlocomotion tracking software (Berghmans, S., Hunt, J., Roach, A., &Goldsmith, P., Zebrafish offer the potential for a primary screen toidentify a wide variety of potential anticonvulsants. Epilepsy Res. 75,18-28 (2007); Baxendale, S., et al., Identification of compounds withanti-convulsant properties in a zebrafish model of epileptic seizures.Dis. Model. Mech. 5, 773-774 (2012); Winter, M. J., et al., Validationof a larval zebrafish locomotor assay for assessing the seizureliability of early-stage development drugs. J. Pharm. Tox. Methods 5,176-187 (2008)). Seizures in scn1Lab zebrafish mutants were responsiveto the ketogenic diet and four AEDs (e.g., valproate, benzodiazepine,potassium bromide and stiripentol) prescribed clinically for patientswith DS.

Interestingly, electrographic seizure events in scn1Lab mutants remainedunchanged (or perhaps worsened) in response to several commerciallyavailable AEDs. While it is possible that drug concentrations higherthan 1 mM could be required to abolish electrical events, these would beconsidered high and potentially non-selective concentrations. In drugtrials using an acute PTZ-induced seizure model in larval zebrafish(Baraban, S. C., et al., A large-scale mutagenesis screen to identifyseizure-resistant zebrafish. Epilepsia 48, 1151-157 (2007); Berghmans,S., Hunt, J., Roach, A., & Goldsmith, P., Zebrafish offer the potentialfor a primary screen to identify a wide variety of potentialanticonvulsants. Epilepsy Res. 75, 18-28 (2007); Baxendale, S., et al.,Identification of compounds with anti-convulsant properties in azebrafish model of epileptic seizures. Dis. Model. Mech. 5, 773-774(2012); Afrikanova, T., et al., Validation of the zebrafishpentylenetetrazol seizure model: locomotor versus electrographicresponses to antiepileptic drugs. PLoS One 8, e54166 (2013)), AEDconcentrations of 1 mM and below were often sufficient for assessingantiepileptic activity. With a failure to respond to seven differentAEDs this model fits the clinical definition of drug-resistant epilepsy(de Toffol, B., et al., ESPERA study: Applicability of the new ILAEcriteria for antiepileptic drug resistance of focal epilepsies incurrent clinical practice. Epilepsy Beh 25, 166-169 (2012)).

For nearly 40 years, the discovery and identification of new AEDs hasalmost entirely been based upon preclinical animal models of acquired oracute seizures in rodents (Loscher, W. & Schmidt, D., Modernantiepileptic drug development has failed to deliver: Ways out of thecurrent dilemma. Epilepsia 52, 657-658 (2011)). This approachsuccessfully identified drugs that block generalized tonic-clonicseizures in humans (Bialer, M. & White H. S., Key factors in thediscovery and development of new antiepileptic drugs. Nat. Rev. DrugDiscov. 9, 10-19 (2012)) but remains time-consuming, resource intensive,expensive and laborious. While testing against PTZ or other types ofacquired seizures in zebrafish larvae may be more efficient than similarassays in rodents (Berghmans, S., Hunt, J., Roach, A., & Goldsmith, P.,Zebrafish offer the potential for a primary screen to identify a widevariety of potential anticonvulsants. Epilepsy Res. 75, 18-28 (2007);Baxendale, S., et al., Identification of compounds with anti-convulsantproperties in a zebrafish model of epileptic seizures. Dis. Model. Mech.5, 773-774 (2012) Afrikanova, T., et al., Validation of the zebrafishpentylenetetrazol seizure model: locomotor versus electrographicresponses to antiepileptic drugs. PLoS One 8, e54166 (2013)), theyultimately should identify the same classes of compounds.

In contrast, here is described an alternative screening strategy using a96-well format for rapid automated behavioral monitoring followed by asensitive electrophysiological assay of spontaneous electrographicseizure activity in a mutant fish mimicking a known human geneticdisorder. This in vivo strategy simultaneously monitors lethality and isnot limited to SCN1A, but could be applied to any epilepsy disorder.Indeed, this phenotype-based approach could form the basis of agenetically informed or “personalized” approach to drug discovery. Whilegenetically modified mice mimicking known SCN1A mutations and exhibitingepilepsy have been developed, breeding can be complicated, backgroundstrain can modify seizure phenotypes and AEDs are rarely tested in theseanimals. For example, in Scn1a^(RX/+) mutant mice stiripentol andclobazam were only evaluated for effects on hyperthermia-induced seizurethresholds (Cao, D., et al., Efficacy of stiripentol inhyperthermia-induced seizures in a mouse model of Dravet syndrome.Epilepsia 53, 1140-1145 (2012)). Treatment of Scn1a^(+/−) mutant micewith clonazepam, an allosteric modulator of GABA-A receptors, rescuedsome of the autistic-like behaviors but was not evaluated as anantiepileptic (de Toffol, B., et al., ESPERA study: Applicability of thenew ILAE criteria for antiepileptic drug resistance of focal epilepsiesin current clinical practice. Epilepsy Beh 25, 166-169 (2012)).

Where drug-resistant rodent epilepsy models have been described, such asthe subgroup of wild-type rats selected from kindling or post-statusepilepticus models (Han, S., et al., Autistic-like behaviour in Scn1a+/−mice and rescue by enhanced GABA-mediated neurotransmission. Nature 489,385-390 (2012)), they remain only poorly characterized and are notsuitable to initial high-throughput stages of drug screening. Incontrast, using a zebrafish scn1Lab mutant with greater than 75%sequence identity for a human sodium channel mutation, a large-scaletranscriptomic profiling of over 44,000 probes was completed,demonstrated a developmental progression of scn1Lab channel expressionand epileptic phenotypes, analyzed the effects of availableantiepileptic therapies, and screened a 320 compound chemical libraryagainst spontaneous unprovoked seizures. Although this firstproof-of-principle screen was accomplished at one fish per well, 6 to 12fish per trial and one trial per week, the ease with which zebrafishcould be scaled upward (especially in a commercial setting) to studyhundreds to thousands of larvae per week make this an attractive systemfor a rapid large-scale first-stage in vivo drug discovery program.Simultaneous in vivo evaluation of toxicity—one of the greatest sourcesof failure in moving lead compounds from the bench to the clinic—is acritical advantage of this approach over available organotypichippocampal culture- or in silica-based screening strategies.

Although any animal model drug discovery data should be treatedcautiously, clemizole, a compound with H1 antagonist and NS4B RNAinhibiting properties, is an FDA-approved drug with a safe toxicologyprofile emerged from this screen and offers an exciting starting pointfor further research. For example, although it was recently recognizedthat antihistamines inhibit induced seizures in neonatal rats (Yamada,K., Takizawa, F., Tamura, T., & Kanda T., The effect of antihistamineson seizures induced by increasing-current electroshocks: ketotifen, butnot olopatadine, promotes the seizures in infant rats. Biol. Pharm.Bull. 35, 693-697 (2012)), without being bound by any particular theory,this is likely not the mechanism of action here. We demonstrated fourother H1 antihistamines (pimethixene maleate, chloropyramine HCl,mebhydrolin napthalenesulfonate and iproheptine) failed to suppressconvulsive behavior in scn1Lab mutants. Furthermore, evidence suggeststhe potential for H1 antihistamines to adversely modify seizures inchildren (Miyata, I., Saegusa, H., & Sakurai, M., Seizure-modifyingpotential of histamine H1 antagonists: a clinical observation. Pediatr.Int. 53, 706-708 (2011)) indicating that more detailed analysis will berequired to identify a mechanism of action. Given that clemizole wasalso effective in a zebrafish version of the Metrazol test it may beworthwhile to pursue additional pre-clinical testing in theNIH-sponsored Anticonvulsant Drug Development Program at the Universityof Utah. Most importantly, these studies suggest that in vivo drugscreening and experimental analysis of scn1Lab mutant zebrafish couldprove extremely valuable to understanding (and treatment) of Dravetsyndrome.

Animals. Scn1Lab (didy^(s552)) zebrafish embryos were a kind gift fromHerwig Baier. Adult HuC:GFP zebrafish were a kind gift from StephenEkker. Zebrafish were generated and maintained in accordance with theguidelines of the University of California, San Francisco Committee onthe Use and Care of Animals. Zebrafish larvae were maintained in “embryomedium” consisting of 0.03% Instant Ocean (Aquarium Systems, Inc.,Mentor, Ohio, U.S.A.) in deionized water containing 0.002% MethyleneBlue as a fungicide. Larval zebrafish clutches were bred from scn1Labheterozygous animals that had been backcrossed to TL wild-type orHuC:GFP zebrafish for at least 7 generations. Homozygous mutants (sortedbased on pigmentation) and age-matched sibling larvae were used.Although the precise genetic defect responsible for the skinpigmentation issue is unknown, it is interesting that a 1.5-foldup-regulation of a gene encoding the melanocortin 5a receptor was notedin the microarray data.

Seizure monitoring. Procedures for locomotion tracking andelectrophysiology were described (Baraban, S. C., et al., A large-scalemutagenesis screen to identify seizure-resistant zebrafish. Epilepsia48, 1151-157 (2007); Baraban, S. C., Taylor, M. R., Castro, P. A., &Baier H., Pentylenetetrazole induced changes in zebrafish behavior,neural activity and c-fos expression. Neuroscience 131, 759-768 (2005)).In pilot experiments, HuC:GFP zebrafish were used in electrophysiologyexperiments to obtain an estimation of the location of recordingelectrodes. Locomotion plots were obtained for one fish per well at arecording epoch of 10 min using a DanioVision system running EthoVisionXT software (Noldus Information Technology; Leesburg, Va.). Seizurescoring was performed as described (Baraban, S. C., Taylor, M. R.,Castro, P. A., & Baier H., Pentylenetetrazole induced changes inzebrafish behavior, neural activity and c-fos expression. Neuroscience131, 759-768 (2005)). Locomotion plots were analyzed for distancetraveled (in mm) and mean velocity (in mm/sec). Epileptiform events wereanalyzed in pClamp (Molecular Devices; Sunnyvale, Calif.) and defined asupward or downward membrane deflections greater than 2× baseline noiselevel and classified as either interictal-like (100 to 300 msecduration) or ictal-like (1000 to 5000 msec duration). Burst frequencywas determined by counting the number of epileptiform events per minuteduring a 10-min recording epoch. Burst duration was determined bymeasuring the onset-to-offset interval for all events during the sameepoch.

Drugs were obtained from Sigma-Aldrich and dissolved in embryo media.Stock solutions were prepared in embryo media at 1 mM and pH adjusted to˜7.5. Ganaxolone was a kind gift from BioCrea GmbH (Radebeul, Germany).Compounds for drug screening were purchased from MicroSource DiscoverySystems, Inc. (International Drug Collection; Gaylordsville, Conn.) andwere provided as 10 mM DMSO solutions. Test compounds were dissolved inembryo media and tested at concentrations between 6.7 and 667 μM; finalDMSO concentration ˜7%. An initial screen concentration of 667 μM waschosen for behavioral studies in freely swimming fish as this falls onthe lower range of AED concentrations previously reported in to beeffective against PTZ (10-20 mM) induced seizures in larval zebrafish(0.1 to 25 mM) Baraban, S. C., Taylor, M. R., Castro, P. A., & Baier H.,Pentylenetetrazole induced changes in zebrafish behavior, neuralactivity and c-fos expression. Neuroscience 131, 759-768 (2005);Berghmans, S., Hunt, J., Roach, A., & Goldsmith, P., Zebrafish offer thepotential for a primary screen to identify a wide variety of potentialanticonvulsants. Epilepsy Res. 75, 18-28 (2007); Afrikanova, T., et al.,Validation of the zebrafish pentylenetetrazol seizure model: locomotorversus electrographic responses to antiepileptic drugs. PLoS One 8,e54166 (2013)) and was the most efficient use of the small volume ofstock solution (250 μL) provided by MicroSource Discovery Systems, Inc.A slightly higher concentration (1 mM) was chosen for the initial AEDvalidation assays in FIGS. 5 and 6 to account for any potentialcomplications associated with diffusion through the agar. DMSO wasevaluated for toxicity at dilutions between 0.01 and 100% usingwild-type larvae (n=12 fish per concentration); DMSO at >25% was lethal.

In all drug screening studies compounds were coded and experiments wereperformed by investigators blind to the nature of the compound. Baselinerecordings of seizure activity were obtained from mutants bathed inembryo media; a second plot was then obtained following a solutionchange to a test compound. Each test compound classified as a “positivehit” in the locomotion assay was visually confirmed as alive based onmovement in response to touch and visible heartbeat. WT fish exhibitlittle to no spontaneous swim activity during these 10 min recordingepochs (see FIG. 3B) and were not used in the drug discovery assay.

Procedures for microarray, quantitative PCR and whole-mount in situhybridization were described (Hortopan, G. A., et al., Spontaneousseizures and altered gene expression in GABA signaling pathways in amind bomb mutant zebrafish. J. Neurosci. 30, 13718-13728 (2010)).

Data are presented as mean and SEM, unless stated otherwise. Pairwisestatistical significance was determined with Student's two-tailedunpaired t-test, ANOVA or Mann-Whitney rank sum test, as appropriate,unless stated otherwise. Results were considered significant at P<0.05,unless otherwise indicated.

Example 2

Based on our previous discussion and some activity observed at the 100and 300 mg/kg doses in the qualitative MES screen in mice, we proceededwith quantitative testing in the MES/scMET/Tox mouse model to determinethe ED50/TD50. During the determination of the TPE in the MES model, noactivity was observed at the 300 mg/kg starting dose. However, activitywas observed at the 500 mg/kg dose with 2/4 animals protected at 0.25min and 4/4 animals protected at 30 minutes. No activity or toxicity(unable to grasp rotorod) was observed at any other dose or time pointtested. No activity was observed in the scMET model. The data in the MESmodel shows that there is significant activity/protection with ASP469016in this mouse model with an ED50<400 mg/kg.

Example 3

ASP469016 was tested in our initial T31 (MES/scMET/Tox) screen at 30,100, and 300 mg/kg. The data for each condition is presented as N/F,where N equals the number of animals protected and F equals the numberof animals tested. For tests of toxicity (TOX), N equals the number ofanimals displaying toxic effects and F equals the number of animalstested. Codes in the C column refer to comments from the techniciansperforming the experiment and are defined in the comments section ifnecessary. Any deaths are noted. As shown in the 6 Hz (32 mA) model only¼ animals were protected at 100 mg/kg at 30 min. In the MES-inducedseizure model only ¼ animals were protected at 100 and 300 mg/kg at 30min. No toxicity (unable to grasp rotorod) or activity was detected atany other dose or time point tested.

Anticonvulsant Screening Results—Mice MES and 6 Hz Identification

Example 4

Clemizole (149934-L6) was tested in the CEREP BioPrint Profile, which isa panel of 139 different in vitro receptor binding and enzyme assays.For the initial BioPrint screen, a free compound concentration of 10 μM(1.0E-5 M) was used. Compound binding was calculated as a % inhibitionof the binding of a radioactively labeled ligand specific for eachtarget. Compound enzyme inhibition effect was calculated as a %inhibition of control enzyme activity. In each experiment, therespective reference compound was tested concurrently with Clemizole(149934-L6), and the data were compared with historical valuesdetermined at CEREP. The experiment was accepted in accordance withCEREP's validation Standard Operating Procedure. Results showing aninhibition (or stimulation for assays run in basal conditions) higherthan 50% are considered to represent significant effects of the testcompounds. A summary of these results is shown below:

Assay 1.0E-05M 5-HT_(2A)(h) (agonist radioligand) 86% 5-HT_(2B)(h)(agonist radioligand) 82.5% 5-HT_(1A)(h) (agonist radioligand) 19.5%5-HT_(1B)(h) (agonist radioligand) 4.4% 5-HT_(1D)(h) (agonistradioligand) 22.7% 5-HT₃(h) (agonist radioligand) 9.8% 5-HT_(4e)(h)(agonist radioligand) −1.0% GABA_(A1)(h) (agonist radioligand) −9.6%GABA_(B(1b))(h) (agonist radioligand) −5.9% BZD (central) (agonistradioligand) −19.6% CB₁(h) (agonist radioligand) 17.9% CB₂(h) (agonistradioligand) 28.4% GABA-gated Cl channel 24.9% SK-Ca channel −0.2% GABAtransporter −5.9%

Example 5

Clemizole does not exert antiepileptic activity via ananti-histaminergic mechanism of action. 32 different antihistaminecompounds, FIG. 10, were screened in the scn1Lab zebrafish assay andnone of the compounds mimicked the antiepileptic action of clemizole.Three of the compounds were toxic and, consistent with clinical reportsthat antihistamines can exacerbate seizures in pediatric epilepsypatients, five compounds increased seizure behavior.

Example 6

Applicants screened a Selleck Customized Library containing 62 drugsacting on serotonin signaling pathways. These compounds were initiallyscreened in the zebrafish locomotion assay and 15 compounds wereidentified as positive hits in the first-pass locomotion assay (detailsof the assay can be found in Baraban et al. Nat. Comm. 2013 and Dindayand Baraban, eNeuro 2015) and can be seen in FIG. 11. These studiessuggest that modulation of 5HT signaling, especially activation ofpostsynaptic 5HT receptors, has potential antiepileptic activity.

Concentration-response re-test studies on all of the 5HT compoundsidentified in the first-pass locomotion assay. Trazodone results(Desryl, Oleptro) is shown as a representative example. In addition toshowing robust inhibition of spontaneous seizure behavior in thelocomotion assay at concentrations between 100 and 750 μM (FIG. 12A),trazodone also effectively suppressed EEG activity in the scn1Labmutants (n=15) at concentrations between 250 and 500 μM (FIG. 12B) andin separate trials with drug washout (n=12). Compounds with positivehits include sumatriptan, naratriptap, rizatriptan, zolmitriptan,urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol),lorcaserin, buspirone, ziprasidone, TCB-2((4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide),BRL-15572(3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol),trazodone, BMY 7378(8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione),atomoxetine, and venlafaxine.

ADDITIONAL EMBODIMENTS

Embodiment 1. A method of treating an epilepsy disorder, the methodincluding administering to a subject in need thereof a therapeuticallyeffective amount of a 5HT receptor agonist, or a pharmaceuticallyacceptable salt thereof.

Embodiment 2. The method of embodiment 1, wherein the 5HT receptoragonist is a 5HT2A receptor agonist or a 5HT2B receptor agonist.

Embodiment 3. The method of embodiment 2, wherein the 5HT receptoragonist is a dual 5HT2A receptor and 5HT2B receptor agonist.

Embodiment 4. The method of embodiment 1, wherein the 5HT receptoragonist is not clemizole or fenfluramine.

Embodiment 5. The method of embodiment 1, wherein the 5HT receptoragonist binds directly to a 5HT receptor.

Embodiment 6. The method of embodiment 1, wherein the 5HT receptoragonist specifically activates a 5HT receptor.

Embodiment 7. The method of embodiment 1, wherein the 5HT receptoragonist provides increased activity mediated by 5HT2A receptor or 5HT2Breceptor with equivalent or reduced activity mediated through 5HT2Creceptor.

Embodiment 8. The method of embodiment 1, wherein the 5HT receptoragonist is not a serotonin reuptake inhibitor.

Embodiment 9. The method of embodiment 1, wherein the 5HT receptoragonist does not significantly bind to or modulate the activity of atleast one of 5HT1A, 5HT1B, 5HT1D, 5HT2C, 5HT3, 5HT4, 5HT6, 5HT7, NPY Y1receptor, L-type Ca channel, N-type Ca channel, SK-Ca channel,GABA-gated C1 channel, GABA transporter, GABA-A1 receptor, GABA-B1breceptor, Na channel, 5HT transporter, CB1 receptor, CB2 receptor, BZDor Estrogen ER alpha.

Embodiment 10. The method of embodiment 1, wherein the 5HT receptoragonist is flibanserin, DOI HCl, norfenfluramine or BW 723C86.

Embodiment 11. The method of embodiment 1, wherein the 5HT receptoragonist is sumatriptan, naratriptap, rizatriptan, zolmitriptan,urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol),lorcaserin, buspirone, ziprasidone, TCB-2((4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide),BRL-15572(3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol),trazodone, BMY 7378(8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione),atomoxetine, or venlafaxine.

Embodiment 12. The method of embodiment 1, wherein the 5HT receptoragonist is trazodone or a pharmaceutically acceptable salt thereof. Inembodiments, the pharmaceutically acceptable salt of trazadone ishydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate,nitrate, maleate, acetate, citrate, fumarate, propionate, tartrate,succinate, benzoate, glutamate, or a quaternary ammonium salt.

Embodiment 13. The method of embodiment 1, wherein the epilepsy disorderis Dravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, orOhtahara Syndrome.

Embodiment 14. The method of embodiment 13, wherein the epilepsydisorder is Dravet Syndrome.

Embodiment 15. The method of embodiment 1, wherein the epilepsy disorderis a pediatric epilepsy disorder.

Embodiment 16. The method of embodiment 1, wherein the subject has acardiovascular disease.

Embodiment 17. The method of embodiment 1, wherein the subject isresistant to treatment with a serotonin reuptake inhibitor.

Embodiment 18. The method of embodiment 1, wherein the subject issusceptible to side effects when administered a serotonin reuptakeinhibitor.

Embodiment 19. The method of embodiment 17 or 18 or, wherein theserotonin reuptake inhibitor is fenfluramine.

Embodiment 20. The method of embodiment 1, wherein the subject has aketogenic diet.

Embodiment 21. The method of embodiment 1, wherein the 5HT receptoragonist inhibits compulsive behaviors or electrographic seizures in anepilepsy subject, an Alzheimer's disease subject, an autism subject, ora Parkinson's disease subject.

Embodiment 22. The method of embodiment 1, wherein the 5HT receptoragonist reduces the incidence of unprovoked seizures in the subject whencompared to the absence of the 5HT receptor agonist.

Embodiment 23. The method of embodiment 1, wherein the administration ofthe 5HT receptor agonist reduces or prevents myoclonus seizures orstatus epilepticus in the subject when compared to the absence of 5HTreceptor agonist.

Embodiment 24. The method of embodiment 1, wherein the 5HT receptoragonist is administered to the subject at an amount of about 0.1 mg toabout 1000 mg per kg body weight.

Embodiment 25. The method of embodiment 22, wherein the 5HT receptoragonist is administered to the subject in a daily dose of about 0.1 mgto about 1000 mg per kg body weight to the subject.

Embodiment 26. The method of embodiment 1, wherein the 5HT receptoragonist is co-administered with an anti-epileptic drug (AED).

Embodiment 27. The method of embodiment 1, wherein the 5HT receptoragonist is an adjunctive therapy with an anti-epileptic drug (AED).

Embodiment 28. The method of embodiment 26 or 27, wherein the AED isacetazolamide, benzodiazepine, cannabadiols, carbamazepine, clobazam,clonazepam, eslicarbazepine acetate, ethosuximide, ethotoin, felbamate,fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A,lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine,perampanel, piracetam, phenobarbital, phenytoin, potassium bromide,pregabalin, primidone, retigabine, rufinamide, valproic acid, sodiumvalproate, stiripentol, tiagabine, topiramate, vigabatrin, orzonisamide.

Embodiment 29. The method of embodiment 28, wherein the AED is valproicacid, sodium valproate, clonazepam, ethosuximide, felbamate, gabapentin,carbamazepine, oxcarbazepine, lamotrigine, levetiracetam,benzodiazepine, phenobarbital, pregabalin, primidone, tiagabine,topiramate, potassium bromide, phenytoin, stiripentol, vigabatrin, orzonisamide.

Embodiment 30. The method of embodiment 29, wherein the AED is valproicacid, sodium valproate, gabapentin, topiramate, carbamazepine,oxcarbazepine, or vigabatrin.

Embodiment 31. The method of embodiment 26, wherein the AED is notfenfluramine or topiramate.

Embodiment 32. The method of embodiment 26, wherein the AED isadministered simultaneously with or sequentially with the 5HT receptoragonist.

Embodiment 33. A method of treating an epilepsy disorder, the methodincluding administering to a subject in need thereof a therapeuticallyeffective amount of a 5HT receptor agonist, or a pharmaceuticallyacceptable salt thereof, wherein the subject has a cardiovasculardisease, is resistant to treatment with a serotonin reuptake inhibitor,or is susceptible to side effects when administered a serotonin reuptakeinhibitor.

Embodiment 34. The method of embodiment 33, wherein the 5HT receptoragonist is a 5HT2A receptor agonist or a 5HT2B receptor agonist.

Embodiment 35. The method of embodiment 32, wherein the 5HT receptoragonist is a dual 5HT2A receptor and 5HT2B receptor agonist.

Embodiment 36. The method of embodiment 33, wherein the 5HT receptoragonist is clemizole, a clemizole analog, or a pharmaceuticallyacceptable salt thereof.

Embodiment 37. The method of embodiment 36, wherein the pharmaceuticallyacceptable salt is clemizole HCl.

Embodiment 38. The method of embodiment 36, wherein the clemizole, theclemizole analog, or the pharmaceutically acceptable salt thereof formspart of a pharmaceutical composition.

Embodiment 39. The method of embodiment 38, wherein the pharmaceuticalcomposition further includes a pharmaceutically acceptable excipient.

Embodiment 40. The method of embodiment 38, wherein the pharmaceuticalcomposition includes a therapeutically effective amount of clemizole,the clemizole analog, or the pharmaceutically acceptable salt thereof.

Embodiment 41. The method of embodiment 40, wherein the pharmaceuticalcomposition is co-administered with an anti-epileptic drug (AED).

Embodiment 42. The method of embodiment 41, wherein the pharmaceuticalcomposition includes clemizole, the clemizole analog, or thepharmaceutically acceptable salt thereof and an AED.

Embodiment 43. The method of embodiment 33, wherein the 5HT receptoragonist is not fenfluramine.

Embodiment 44. The method of embodiment 33, wherein the 5HT receptoragonist binds directly to a 5HT receptor.

Embodiment 45. The method of embodiment 33, wherein the 5HT receptoragonist specifically activates a 5HT receptor.

Embodiment 46. The method of embodiment 33, wherein the 5HT receptoragonist provides increased activity mediated by 5HT2A receptor or 5HT2Breceptor with equivalent or reduced activity mediated through 5HT2Creceptor.

Embodiment 47. The method of embodiment 33, wherein the 5HT receptoragonist is not a serotonin reuptake inhibitor.

Embodiment 48. The method of embodiment 33, wherein the 5HT receptoragonist does not significantly bind to or modulate the activity of atleast one of 5HT1A, 5HT1B, 5HT1D, 5HT2C, 5HT3, 5HT4, 5HT6, 5HT7, NPY Y1receptor, L-type Ca channel, N-type Ca channel, SK-Ca channel,GABA-gated C1 channel, GABA transporter, GABA-A1 receptor, GABA-B1breceptor, Na channel, 5HT transporter, CB1 receptor, CB2 receptor, BZDor Estrogen ER alpha.

Embodiment 49. The method of embodiment 33, wherein the 5HT receptoragonist is sumatriptan, naratriptap, rizatriptan, zolmitriptan,urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol),lorcaserin, buspirone, ziprasidone, TCB-2((4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide),BRL-15572(3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol),trazodone, BMY 7378(8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione),atomoxetine, or venlafaxine.

Embodiment 50. The method of embodiment 33, wherein the 5HT receptoragonist is trazodone or a pharmaceutically acceptable salt thereof.

Embodiment 51. A method of modulating activity of a 5HT receptorincluding contacting a 5HT receptor with clemizole, a clemizole analog,or a pharmaceutically acceptable salt thereof.

Embodiment 52. The method of embodiment 51, wherein the modulating isactivating.

Embodiment 53. The method of embodiment 51, wherein the 5HT receptor isa 5HT2A receptor or a 5HT2B receptor.

Embodiment 54. A method of treating a disease or disorder caused by adeficiency of serotonin in the brain or under activity of one or more5HT receptors including administering to a subject in need thereof atherapeutically effective amount of clemizole, a clemizole analog, or apharmaceutically acceptable salt thereof.

Embodiment 55. The method of embodiment 54, wherein the disease ordisorder is not epilepsy.

Embodiment 56. The method of embodiment 54, wherein the disease ordisorder is not Dravet Syndrome.

Embodiment 57. The method of embodiment 51, wherein the disease ordisorder is selected from the group consisting of migraine, Fragile Xsyndrome, Prader-Willi syndrome, schizophrenia, depression, Alzheimer'sdisease, autism, neuropathic pain, Parkinson's disease, irritable bowel,disorder, and dementia.

Embodiment 58. The method of embodiment 50, wherein the pharmaceuticallyacceptable salt is clemizole HCl.

What is claimed is:
 1. A method of treating an epilepsy disorder, said method comprising administering to a subject in need thereof a therapeutically effective amount of a 5HT receptor agonist, or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein said 5HT receptor agonist is a 5HT_(2A) receptor agonist or a 5HT_(2B) receptor agonist.
 3. The method of claim 2, wherein the 5HT receptor agonist is a dual 5HT_(2A) receptor and 5HT_(2B) receptor agonist.
 4. The method of claim 1, wherein said 5HT receptor agonist is not clemizole or fenfluramine.
 5. The method of claim 1, wherein said 5HT receptor agonist binds directly to a 5HT receptor.
 6. The method of claim 1, wherein said 5HT receptor agonist specifically activates a 5HT receptor.
 7. The method of claim 1, wherein said 5HT receptor agonist provides increased activity mediated by 5HT_(2A) receptor or 5HT_(2B) receptor with equivalent or reduced activity mediated through 5HT_(2C) receptor.
 8. The method of claim 1, wherein said 5HT receptor agonist is not a serotonin reuptake inhibitor.
 9. The method of claim 1, wherein said 5HT receptor agonist does not significantly bind to or modulate the activity of at least one of 5HT_(1A), 5HT_(1B), 5HT_(1D), 5HT_(2C), 5HT₃, 5HT₄, 5HT₆, 5HT₇, NPY Y1 receptor, L-type Ca channel, N-type Ca channel, SK-Ca channel, GABA-gated C1 channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Na channel, 5HT transporter, CB1 receptor, CB2 receptor, BZD or Estrogen ER alpha.
 10. The method of claim 1, wherein said 5HT receptor agonist is flibanserin, DOI HCl, norfenfluramine or BW 723C86.
 11. The method of claim 1, wherein said 5HT receptor agonist is sumatriptan, naratriptap, rizatriptan, zolmitriptan, urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol),lorcaserin, buspirone, ziprasidone, TCB-2 ((4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide), BRL-15572 (3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol), trazodone, BMY 7378 (8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione), atomoxetine, or venlafaxine.
 12. The method of claim 1, wherein said 5HT receptor agonist is trazodone.
 13. The method of claim 1, wherein said epilepsy disorder is Dravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, or Ohtahara Syndrome.
 14. The method of claim 13, wherein said epilepsy disorder is Dravet Syndrome.
 15. The method of claim 1, wherein said epilepsy disorder is a pediatric epilepsy disorder.
 16. The method of claim 1, wherein said subject has a cardiovascular disease.
 17. The method of claim 1, wherein said subject is resistant to treatment with a serotonin reuptake inhibitor.
 18. The method of claim 1, wherein said subject is susceptible to side effects when administered a serotonin reuptake inhibitor.
 19. The method of claim 17 or 18 or, wherein said serotonin reuptake inhibitor is fenfluramine.
 20. The method of claim 1, wherein said subject has a ketogenic diet.
 21. The method of claim 1, wherein said 5HT receptor agonist inhibits compulsive behaviors or electrographic seizures in an epilepsy subject, an Alzheimer's disease subject, an autism subject, or a Parkinson's disease subject.
 22. The method of claim 1, wherein said 5HT receptor agonist reduces the incidence of unprovoked seizures in said subject when compared to the absence of said 5HT receptor agonist.
 23. The method of claim 1, wherein said administration of said 5HT receptor agonist reduces or prevents myoclonus seizures or status epilepticus in said subject when compared to the absence of 5HT receptor agonist.
 24. The method of claim 1, wherein said 5HT receptor agonist is administered to said subject at an amount of about 0.1 mg to about 1000 mg per kg body weight.
 25. The method of claim 22, wherein said 5HT receptor agonist is administered to said subject in a daily dose of about 0.1 mg to about 1000 mg per kg body weight to said subject.
 26. The method of claim 1, wherein said 5HT receptor agonist is co-administered with an anti-epileptic drug (AED).
 27. The method of claim 1, wherein said 5HT receptor agonist is an adjunctive therapy with an anti-epileptic drug (AED).
 28. The method of claim 26 or 27, wherein said AED is acetazolamide, benzodiazepine, cannabadiols, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, ethotoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, potassium bromide, pregabalin, primidone, retigabine, rufinamide, valproic acid, sodium valproate, stiripentol, tiagabine, topiramate, vigabatrin, or zonisamide.
 29. The method of claim 28, wherein said AED is valproic acid, sodium valproate, clonazepam, ethosuximide, felbamate, gabapentin, carbamazepine, oxcarbazepine, lamotrigine, levetiracetam, benzodiazepine, phenobarbital, pregabalin, primidone, tiagabine, topiramate, potassium bromide, phenytoin, stiripentol, vigabatrin, or zonisamide.
 30. The method of claim 29, wherein said AED is valproic acid, sodium valproate, gabapentin, topiramate, carbamazepine, oxcarbazepine, or vigabatrin.
 31. The method of claim 26, wherein said AED is not fenfluramine or topiramate.
 32. The method of claim 26, wherein said AED is administered simultaneously with or sequentially with said 5HT receptor agonist.
 33. A method of treating an epilepsy disorder, said method comprising administering to a subject in need thereof a therapeutically effective amount of a 5HT receptor agonist, or a pharmaceutically acceptable salt thereof, wherein said subject has a cardiovascular disease, is resistant to treatment with a serotonin reuptake inhibitor, or is susceptible to side effects when administered a serotonin reuptake inhibitor.
 34. The method of claim 33, wherein said 5HT receptor agonist is a 5HT_(2A) receptor agonist or a 5HT_(2B) receptor agonist.
 35. The method of claim 32, wherein the 5HT receptor agonist is a dual 5HT_(2A) receptor and 5HT_(2B) receptor agonist.
 36. The method of claim 33, wherein said 5HT receptor agonist is clemizole, a clemizole analog, or a pharmaceutically acceptable salt thereof.
 37. The method of claim 36, wherein said pharmaceutically acceptable salt is clemizole HCl.
 38. The method of claim 36, wherein said clemizole, said clemizole analog, or said pharmaceutically acceptable salt thereof forms part of a pharmaceutical composition.
 39. The method of claim 38, wherein said pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
 40. The method of claim 38, wherein said pharmaceutical composition comprises a therapeutically effective amount of clemizole, said clemizole analog, or said pharmaceutically acceptable salt thereof.
 41. The method of claim 40, wherein said pharmaceutical composition is co-administered with an anti-epileptic drug (AED).
 42. The method of claim 41, wherein said pharmaceutical composition comprises clemizole, said clemizole analog, or said pharmaceutically acceptable salt thereof and an AED.
 43. The method of claim 33, wherein said 5HT receptor agonist is not fenfluramine.
 44. The method of claim 33, wherein said 5HT receptor agonist binds directly to a 5HT receptor.
 45. The method of claim 33, wherein said 5HT receptor agonist specifically activates a 5HT receptor.
 46. The method of claim 33, wherein said 5HT receptor agonist provides increased activity mediated by 5HT_(2A) receptor or 5HT_(2B) receptor with equivalent or reduced activity mediated through 5HT_(2C) receptor.
 47. The method of claim 33, wherein said 5HT receptor agonist is not a serotonin reuptake inhibitor.
 48. The method of claim 33, wherein said 5HT receptor agonist does not significantly bind to or modulate the activity of at least one of 5HT_(1A), 5HT_(1B), 5HT_(1D), 5HT_(2C), 5HT₃, 5HT₄, 5HT₆, 5HT₇, NPY Y1 receptor, L-type Ca channel, N-type Ca channel, SK-Ca channel, GABA-gated C1 channel, GABA transporter, GABA-A1 receptor, GABA-B1b receptor, Na channel, 5HT transporter, CB1 receptor, CB2 receptor, BZD or Estrogen ER alpha.
 49. The method of claim 33, wherein said 5HT receptor agonist is sumatriptan, naratriptap, rizatriptan, zolmitriptan, urapidil, BRL-54443 (3-(1-methylpiperidin-4-yl)-1H-indol-5-ol), lorcaserin, buspirone, ziprasidone, TCB-2 ((4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide), BRL-15572 (3-(4-(4-chlorophenyl)piperazin-1-yl)-1,1-diphenyl-2-propanol), trazodone, BMY 7378 (8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione), atomoxetine, or venlafaxine.
 50. The method of claim 33, wherein said 5HT receptor agonist is trazodone or a pharmaceutically acceptable salt thereof.
 51. A method of modulating activity of a 5HT receptor comprising contacting a 5HT receptor with clemizole, a clemizole analog, or a pharmaceutically acceptable salt thereof.
 52. The method of claim 51, wherein said modulating is activating.
 53. The method of claim 51, wherein said 5HT receptor is a 5HT_(2A) receptor or a 5HT_(2B) receptor.
 54. A method of treating a disease or disorder caused by a deficiency of serotonin in the brain or under activity of one or more 5HT receptors comprising administering to a subject in need thereof a therapeutically effective amount of clemizole, a clemizole analog, or a pharmaceutically acceptable salt thereof.
 55. The method of claim 54, wherein said disease or disorder is not epilepsy.
 56. The method of claim 54, wherein said disease or disorder is not Dravet Syndrome.
 57. The method of claim 51, wherein said disease or disorder is selected from the group consisting of migraine, Fragile X syndrome, Prader-Willi syndrome, schizophrenia, depression, Alzheimer's disease, autism, neuropathic pain, Parkinson's disease, irritable bowel, disorder, and dementia.
 58. The method of claim 50, wherein said pharmaceutically acceptable salt is clemizole HCl. 